Oral immunotherapy tolerizes mice to enzyme replacement therapy for Morquio A syndrome.

Immune response to therapeutic enzymes poses a detriment to patient safety and treatment outcome. Enzyme replacement therapy (ERT) is a standard therapeutic option for some types of mucopolysaccharidoses, including Morquio A syndrome caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Current protocols tolerize patients using cytotoxic immunosuppressives, which can cause adverse effects. Here we show development of tolerance in Morquio A mice via oral delivery of peptide or GALNS for 10 days prior to ERT. Our results show that using an immunodominant peptide (I10) or the complete GALNS enzyme to orally induce tolerance to GALNS prior to ERT resulted in several improvements to ERT in mice: (a) decreased splenocyte proliferation after in vitro GALNS stimulation, (b) modulation of the cytokine secretion profile, (c) decrease in GALNS-specific IgG or IgE in plasma, (d) decreased GAG storage in liver, and (e) fewer circulating immune complexes in plasma. This model could be extrapolated to other lysosomal storage disorders in which immune response hinders ERT.

Peptide-MHC-I from Endogenous Antigen Outnumber Those from Exogenous Antigen, Irrespective of APC Phenotype or Activation.

Naïve anti-viral CD8+ T cells (TCD8+) are activated by the presence of peptide-MHC Class I complexes (pMHC-I) on the surface of professional antigen presenting cells (pAPC). Increasing the number of pMHC-I in vivo can increase the number of responding TCD8+. Antigen can be presented directly or indirectly (cross presentation) from virus-infected and uninfected cells, respectively. Here we determined the relative importance of these two antigen presenting pathways in mousepox, a natural disease of the mouse caused by the poxvirus, ectromelia (ECTV). We demonstrated that ECTV infected several pAPC types (macrophages, B cells, and dendritic cells (DC), including DC subsets), which directly presented pMHC-I to naïve TCD8+ with similar efficiencies in vitro. We also provided evidence that these same cell-types presented antigen in vivo, as they form contacts with antigen-specific TCD8+. Importantly, the number of pMHC-I on infected pAPC (direct presentation) vastly outnumbered those on uninfected cells (cross presentation), where presentation only occurred in a specialized subset of DC. In addition, prior maturation of DC failed to enhance antigen presentation, but markedly inhibited ECTV infection of DC. These results suggest that direct antigen presentation is the dominant pathway in mice during mousepox. In a broader context, these findings indicate that if a virus infects a pAPC then the presentation by that cell is likely to dominate over cross presentation as the most effective mode of generating large quantities of pMHC-I is on the surface of pAPC that endogenously express antigens. Recent trends in vaccine design have focused upon the introduction of exogenous antigens into the MHC Class I processing pathway (cross presentation) in specific pAPC populations. However, use of a pantropic viral vector that targets pAPC to express antigen endogenously likely represents a more effective vaccine strategy than the targeting of exogenous antigen to a limiting pAPC subpopulation.

Autoimmune gastritis mediated by CD4+ T cells promotes the development of gastric cancer.

Chronic inflammation is a major risk factor for cancer, including gastric cancers and other gastrointestinal cancers. For example, chronic inflammation caused by autoimmune gastritis (AIG) is associated with an increased risk of gastric polyps, gastric carcinoid tumors, and possibly adenocarcinomas. In this study, we characterized the progression of gastric cancer in a novel mouse model of AIG. In this model, disease was caused by CD4(+) T cells expressing a transgenic T-cell receptor specific for a peptide from the H(+)/K(+) ATPase proton pump, a protein expressed by parietal cells in the stomach. AIG caused epithelial cell aberrations that mimicked most of those seen in progression of human gastric cancers, including chronic gastritis followed by oxyntic atrophy, mucous neck cell hyperplasia, spasmolytic polypeptide-expressing metaplasia, dysplasia, and ultimately gastric intraepithelial neoplasias. Our work provides the first direct evidence that AIG supports the development of gastric neoplasia and provides a useful model to study how inflammation drives gastric cancer.

Poxvirus interleukin-4 expression overcomes inherent resistance and vaccine-induced immunity: pathogenesis, prophylaxis, and antiviral therapy.

In 2001, Jackson et al. reported that murine IL-4 expression by a recombinant ectromelia virus caused enhanced morbidity and lethality in resistant C57BL/6 mice as well as overcame protective immune memory responses. To achieve a more thorough understanding of this phenomenon and to assess a variety of countermeasures, we constructed a series of ECTV recombinants encoding murine IL-4 under the control of promoters of different strengths and temporal regulation. We showed that the ECTV-IL-4 recombinant expressing the highest level of IL-4 was uniformly lethal for C57BL/6 mice even when previously immunized. The lethality of the ECTV-IL-4 recombinants resulted from virus-expressed IL-4 signaling through the IL-4 receptor but was not due to IL-4 toxicity. A number of treatment approaches were evaluated against the most virulent IL-4 encoding virus. The most efficacious therapy was a combination of two antiviral drugs (CMX001(®) and ST-246(®)) that have different mechanisms of action.

HLA-DRB1 alleles control allergic bronchopulmonary aspergillosis-like pulmonary responses in humanized transgenic mice.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a lung hypersensitivity disease mediated in part by CD4(+) T(H)2 cells. There is a significant association between ABPA and the HLA-DR2 genotypes DRB1(*)1501 and DRB1(*)1503, whereas resistance might be associated with HLA-DRB1(*)1502. OBJECTIVE: We sought to elucidate the role of HLA-DR alleles in allergic inflammation in lungs. METHODS: HLA-DR humanized transgenic mice expressing either the susceptible or resistant alleles were analyzed for the nature and extent of pulmonary inflammation after exposure to Aspergillus species antigens. RESULTS: Exposed DRB1(*)1501 and DRB1(*)1503 transgenic mice displayed infiltrates made up prominently of eosinophils, which is consistent with the inflammation found in ABPA. The resistant DRB1(*)1502 mice, on the other hand, displayed minimal to moderate inflammation, consisting mainly of T-cell infiltrates. Significantly more mucin was produced in the DRB1(*)1503 and DRB1(*)1501 mice, and their ability to limit the number of Aspergillus species conidia within the lung parenchyma was impaired. Despite their differences, both the DRB1(*)1503 and DRB1(*)1502 strains mounted comparable T cell-proliferative responses to Aspergillus species antigens. CONCLUSION: The HLA-DR2 alleles DRB1(*)1501 and DRB1(*)1503 play a major role in the development of allergic pulmonary inflammation. In contrast, the HLA-DR2 allele DRB1(*)1502 mediates a nonallergic T(H)1-like response to the organism, possibly explaining an ABPA resistance factor. These results are in support of our published human studies in patients with cystic fibrosis and asthma. CLINICAL IMPLICATIONS: HLA-DR typing in patients with cystic fibrosis and asthma will aid in the identification of individuals at risk for ABPA.

Antibody responses to vaccinia membrane proteins after smallpox vaccination.

BACKGROUND: Vaccinia virus (VV) membrane proteins are candidates for orthopoxvirus subunit vaccines and potential targets for therapeutic antibodies. Human antibody responses to these proteins after VV vaccination have not been well characterized. METHODS: Pre- and postvaccination (day 26-30) serum specimens from 80 VV vaccine recipients were examined for immunoglobulin G antibodies specific for B5, A33, A27, and L1 by enzyme-linked immunosorbent assay (ELISA). Responses were compared between vaccinia-naive and previously vaccinated (nonnaive) recipients and between nonnaive recipients of undiluted or 1 : 10 diluted vaccine. RESULTS: VV vaccination elicited anti-A33 and anti-A27 antibodies in nearly all vaccinia-naive subjects (100% and 93%, respectively). Preexisting antibodies were commonly detected in nonnaive subjects (for anti-B5, 68%; for anti-A33, 59%; for anti-A27, 38%; and for anti-L1, 10%). Anti-B5 antibodies were strongly boosted by undiluted vaccine (geometric mean titer [GMT], 151 vs. 1010 for pre- vs. postvaccination; P<.001), whereas anti-L1 antibody responses were less robust (detection rate, 31%; GMT, 75) in nonnaive subjects. Diluted vaccine elicited antibody responses that were similar to those elicited by undiluted vaccine. CONCLUSIONS: Vaccination with VV elicits long-lived specific antibody responses directed against VV membrane proteins that vary by previous vaccination status but not with respect to 10-fold dilution of vaccine. B5, A33, and A27 should be considered for inclusion in future human orthopoxvirus subunit vaccines.

Proinflammatory stimuli induce IKKalpha-mediated phosphorylation of PIAS1 to restrict inflammation and immunity.

How inflammatory stimuli signal to the nucleus to restrict inflammation is poorly understood. Protein inhibitor of activated STAT1 (PIAS1), a transcriptional regulator that possesses small ubiquitin-related modifier (SUMO) E3 ligase activity, inhibits immune responses by selectively blocking the binding of NF-kappaB and STAT1 to gene promoters. We report here that PIAS1 becomes rapidly phosphorylated on Ser90 residue in response to various inflammatory stimuli. Mutational studies indicate that Ser90 phosphorylation is required for PIAS1 to repress transcription. Upon TNF treatment, wild-type PIAS1, but not the Ser90A mutant, becomes rapidly associated with the promoters of NF-kappaB target genes. Furthermore, IKKalpha, but not IKKbeta, interacts with PIAS1 in vivo and mediates PIAS1 Ser90 phosphorylation, a process that requires the SUMO ligase activity of PIAS1. Our results identify a signaling pathway in which proinflammatory stimuli activate the IKKalpha-mediated sumoylation-dependent phosphorylation of PIAS1 for the immediate repression of inflammatory gene activation.

MAPK signaling pathways modulate IL-1beta expression in human keratinocytes.

The signaling pathways that modulate IL-1beta expression in human keratinocytes have not been well defined. We have previously shown that TCDD-stimulated AhR-dependent IL-1beta expression in human keratinocytes is due to posttranscriptional regulation involving mRNA stabilization. Since TCDD activates a variety of cellular signaling pathways such as PKC, JNK, and ERK, we investigated these pathways to determine their roles in TCDD-stimulated IL-1beta expression in the human keratinocyte cell line SCC-12F. In this study, we used specific signaling inhibitors to show that ERK and JNK, but not transglutaminase, PKC, or p38, signaling modulate IL-1beta expression. In addition, we show that ERK is constitutively active and unaffected by TCDD treatment and differentiation, while the JNK signaling pathway is modulated by TCDD in an AhR-dependent manner. Thus, both the ERK and JNK MAPK pathways are necessary for IL-1beta expression in TCDD-stimulated human keratinocytes, however, they act at different levels to modulate IL-1beta expression.

Aryl hydrocarbon receptor-mediated posttranscriptional regulation of IL-1beta.

TCDD stimulated IL-1beta gene expression in differentiating human keratinocyte cell lines in a time- and dose-dependent manner. Increases in prointerleukin-1beta (pIL-1beta) protein and IL-1beta steady state mRNA levels were observed in both SCC-12F and HaCaT cells following TCDD treatment. When pretreated with alpha-naphthoflavone, an AhR antagonist, TCDD-mediated increases in IL-1beta gene expression were attenuated, demonstrating for the first time that the environmental toxin, TCDD, can stimulate cytokine (IL-1beta) gene expression in an AhR-dependent manner. Nuclear run-on experiments were performed in SCC-12 cells to determine if the AhR-dependent increases in IL-1beta expression were due to transcriptional activation of the IL-1beta gene. Results showed high constitutive levels of IL-1beta transcriptional activity, however, TCDD treatment, which stimulated IL-1beta steady state mRNA levels, failed to potentiate IL-1beta transcription. Taken together, these results demonstrate that AhR-mediated IL-1beta regulation is occurring posttranscriptionally.

An overview of allergic bronchopulmonary aspergillosis with some new insights.

Allergic bronchopulmonary aspergillosis (ABPA) occurs as a complication of bronchial asthma or cystic fibrosis (CF). The diagnostic criteria speak to an exaggerated type I hypersensitivity response to the ubiquitous organism Aspergillus fumigatus. Immunologic parameters indicative of Aspergillus sensitization in CF may be lost spontaneously. Therefore, it is important that the diagnosis of ABPA in CF include clinical parameters. CF transmembrane regulator gene mutations may occur in asthmatic ABPA patients indicating a subset of ABPA patients that warrant further study to exclude the diagnosis of CF. The extensive tissue damage seen in ABPA may, in part, be caused by proteases released from aspergillus. Host characteristics may predispose to the development of ABPA. It appears that human leukocyte antigen DR2 and particularly DRB1*1503 and *1501 alleles represent significant ABPA susceptibility genes with the possibility that human leukocyte antigen DQ2 allele confers protection in the non-ABPA population. These findings may offer an additional clinical aid in early diagnosis and provide insight into T-cell reactivity in ABPA that may lead to the development of specific immunotherapy.

MHC restriction in allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a rare complication in patients with asthma but more common in patients with cystic fibrosis. In the presence of the fungus Aspergillus fumigatus (Af) in the lower respiratory tract, patients mount a heightened IgG and IgE humoral response specific for Af antigens. Studies on ABPA have suggested a pathogenic role for antigen specific CD4+ Th2 like T lymphocytes producing increased levels of IL-4 and IL-5. MHC class II genes coding for highly polymorphic HLA molecules have been shown to be the likely candidates for controlling immune responses to common allergens. However there has been a lack of information on the pathophysiological role of HLA genes in the development of ABPA. This review describes an association between HLA- class II alleles and the specific responses to Af antigen (Asp f 1) in ABPA. These studies focused on MHC restriction and distribution of HLA- class II alleles in two groups of unrelated North American Caucasian patients with cystic fibrosis and/or asthma. One group consisted of patients with a confirmed diagnosis of ABPA and a second group of patients with Af sensitivity but no ABPA. HLA association studies revealed that the predisposition to develop ABPA is associated with HLA-DR2 and DR5, and possibly DR4 or DR7. A strong association of HLA-DR antigens with ABPA reflects that HLA-DR molecules may present disease-causing peptides. On the other hand a significant association of HLA-DQ2 with Af sensitive nonABPA indicates the involvement of HLA-DQ molecules in protection. A combination of these genetic factors determines the outcome of ABPA in patients with cystic fibrosis and asthma.

T-cell receptor bias in patients with allergic bronchopulmonary aspergillosis.

CD4(+) Th2 helper cell mediated immune responses have been shown to play a crucial role in the pathogenesis of ABPA. HLA and TCR are the candidate genes, which can influence the specificity of these responses. We have previously established a strong association of HLA DR2/5 in ABPA susceptibility. The study was designed to determine whether allergen specific T cell express a limited usage of T cell receptor (TCR) Vbeta gene repertoire in ABPA and to find an association of susceptible HLA-DR determinants with the identified TCR gene segments. TCR Vbeta typing was performed on antigen specific T cell lines from 14 ABPA and 12 nonABPA patients. The majority of ABPA patients (86%) expressed allergen specific T cells with Vbeta13 genes indicating its role in susceptibility, whereas in nonABPA controls, Vbeta1 genes T cell repertoires were predominantly expressed. The unrestricted pattern of Vbeta gene amplification seen before antigen stimulation suggests an oligoclonal expansion of a specific T cell population in response to the allergen Asp f 1 in ABPA and nonABPA patients. The increased usage of Vbeta13 in ABPA and Vbeta1 in nonABPA indicates their importance in susceptibility and resistance, respectively.

Molecular Dissection of the Murine IL-1beta Promoter.

Interleukin-1 (IL-1) is involved in a broad range of biological activities that affect immunological, inflammatory, and nonimmunological responses. Although the role of the IL-1 proteins in normal physiological responses in vivo remains incompletely defined, there is substantial evidence that excessive production of IL-1 contributes to the pathogenesis of many illnesses with autoimmune or inflammatory components, including rheumatoid and osteoarthritis, type I diabetes mellitus and atherosclerosis. Despite numerous reports on IL-1 regulation, very little is known regarding the molecular details of IL-1 production, particularly at the transcription level. This review will focus on our studies of transcriptional regulation of the murine IL-1beta gene and, where appropriate, comparison to similar studies of the human IL-1beta gene. A basic understanding at this level should lead to effective pharmacological intervention and, ultimately, to control of inflammatory disease.