Marco W. J. Schreurs

The spectrum of celiac disease: epidemiology, clinical aspects and treatment

Celiac disease is a gluten-sensitive enteropathy that affects people of all ages worldwide. This disease has emerged as a major health-care problem, as advances in diagnostic and screening methods have revealed its global prevalence. Environmental factors such as gluten introduction at childhood, infectious agents and socioeconomic features, as well as the presence of HLA-DQ2 and/or HLA-DQ8 haplotypes or genetic variations in several non-HLA genes contribute to the development of celiac disease. Growing insight into the variable clinical and histopathological presentation features of this disease has opened new perspectives for future research. A strict life-long gluten-free diet is the only safe and efficient available treatment, yet it results in a social burden. Alternative treatment modalities focus on modification of dietary components, enzymatic degradation of gluten, inhibition of intestinal permeability and modulation of the immune response. A small group of patients with celiac disease (2–5%), however, fail to improve clinically and histologically upon elimination of dietary gluten. This complication is referred to as refractory celiac disease, and imposes a serious risk of developing a virtually lethal enteropathy-associated T-cell lymphoma.

Generation and functional characterization of mouse monocyte-derived dendritic cells.

Dendritic cells (DC) are potent antigen‐presenting cells with the unique capacity to initiate primary immune responses. As a result, DC are currently used in clinical studies to induce immunity against infectious disease and malignant cells. However, multiple DC subsets exist and it has been suggested that the type of DC may affect the immune response induced. The vast majority of DC used in experimental mouse tumor models is derived from bone marrow progenitors. In contrast, most in vitro as well as in vivo human studies involve the use of DC generated from adherent peripheral blood‐derived monocytes in the presence of GM‐CSF and IL‐4. In the current report, we describe for the first time the generation and characterization of mouse monocyte‐derived DC (MODC). The results indicate that mouse MODC display similar morphology, phenotype and immunostimulatory activity as compared to bone marrow‐derived DC. Both DC subsets were able to efficiently take up and subsequently cross‐present protein antigen to cytotoxic T cells. Moreover, we demonstrate that vaccination with peptide‐loaded MODC mediates induction of tumor‐reactive immunity in vivo. The isolation and characterization of mouse MODC will provide a valuable research tool to investigate fundamental aspects of DC biology and which DC subsets are most suitable to induce anti‐tumor immunity.

In vitro priming of tumor-specific cytotoxic T lymphocytes using allogeneic dendritic cells derived from the human MUTZ-3 cell line

The adoptive transfer of in vitro-induced and expanded tumor-specific cytotoxic T lymphocytes (CTL) presents a promising immunotherapeutic approach for the treatment of cancer. The in vitro induction of tumor-reactive CTL requires repeated stimulation of CTL precursors with dendritic cells (DC). To circumvent problems like scarcity of blood DC precursors and donor variability, it would be attractive to use DC from a non-autologous, unlimited source. DCs derived from the human acute myeloid leukemia (AML) cell line MUTZ-3 are attractive candidates since these DCs closely resemble monocyte-derived DC (MoDC) in terms of phenotype and T cell stimulatory capacity. Here we demonstrate that functional CTL clones could be generated against multiple tumor-associated antigens, i.e., human telomerase reverse transcriptase (hTERT), ErbB3-binding protein-1 (Ebp1), carcinoembryonic antigen (CEA) and Her-2/neu, by stimulating CD8β+ CTL precursors with peptide-loaded allogeneic, HLA-A2-matched MUTZ-3-derived DC. A consistent induction capacity, as determined by MHC tetramer-binding, was found in multiple donors and comparable to autologous peptide-loaded MoDC. Functional characterization at the clonal level revealed the priming of CTL that recognized endogenously processed epitopes on tumor cell lines in an HLA-A2-restricted fashion. Our data indicate that MUTZ-3-derived DC can be used as stimulator cells for in vitro priming and expansion of functional TAA-specific effector CTL. MUTZ-3-derived DCs thus represent a ready and standardized source of allogeneic DC to generate CTL for therapeutic adoptive transfer strategies.

Prospective Human Leukocyte Antigen, Endomysium Immunoglobulin A Antibodies, and Transglutaminase Antibodies Testing for Celiac Disease in Children with Down Syndrome

OBJECTIVE To assess the effect of a prospective screening strategy for the early diagnosis of celiac disease (CD) in children with Down syndrome (DS). STUDY DESIGN Blood samples were taken from 155 children with DS. Buccal swabs were also taken from 9 of these children for determination of human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 positivity. Independently, immunoglobulin A anti-endomysium-(EMA) and anti-tissue transglutaminase antibodies (TGA) were tested. An intestinal biopsy was performed to confirm the diagnosis of CD. RESULTS Sixty-three children (40.6%) had test results that were positive for HLA-DQ2 or HLA-DQ8. Results of HLA DQ-typing of DNA isolated from blood and buccal swabs were identical. Eight of the children in whom test results were positive for HLA-DQ2/8 also had positive test results for EMA and TGA. CD was confirmed in 7 of these children with an intestinal biopsy, and in 1 child, CD was suggested with improvement on a gluten-free diet. CONCLUSIONS We found a prevalence of CD in children with DS of 5.2% (10 times higher than the general Dutch population). We recommend HLA-DQ2/8 typing from buccal swabs in the first year of life and initiating serologic screening of children with DS in whom test results are positive for HLA-DQ2 or DQ8 at age 3 years. Early knowledge of negative HLA-DQ2/8 status can reassure most parents that their children do not have a CD risk.

Pharmacological induction of interferon type I activity following treatment with rituximab determines clinical response in rheumatoid arthritis

Objective Despite the fact that rituximab depletes B cells in all treated patients with RA, not all patients show a favourable clinical response. The goal of this study was to provide insight into pharmacological changes in peripheral blood that are associated with clinical response to rituximab. Methods Gene expression profiling was performed on peripheral blood RNA of 13 patients with RA (test group) using Illumina HumanHT beadchip microarrays. An independent group of nine patients was used for validation using TaqMan quantitative PCR. Clinical responder status was determined after 6 months using change in 28-joint Disease Activity Score (ΔDAS28) and European League Against Rheumatism (EULAR) response criteria. Significance analysis of microarrays and ontology analysis were used for data analysis and interpretation. Results Pharmacogenomic analyses demonstrated marked interindividual differences in the pharmacological responses at 3 and 6 months after start of treatment with rituximab. Interestingly, only differences in the regulation of type I interferon (IFN)-response genes after 3 months correlated with the ΔDAS28 response. Good responders (∆DAS>1.2; n=7) exhibited a selective increase in the expression of type I IFN-response genes, whereas this activity was unchanged or hardly changed in non-responders (∆DAS<1.2; n=6) (p=0.0040 at a cut-off of 1.1-fold induction). Similar results were obtained using EULAR response criteria. These results were validated in an independent cohort of nine patients (five non-responders and four responders, p=0.0317). Conclusions A good clinical response to rituximab in RA is associated with a selective drug-induced increase in type I IFN-response activity in patients with RA. This finding may provide insight in the biological mechanism underlying the therapeutic response to rituximab.

Oral DNA vaccination: antigen uptake and presentation by dendritic cells elicits protective immunity

Melanoma differentiation antigens, such as glycoprotein 100 (gp100), have been shown to induce both cellular and humoral immune responses against melanoma in mouse and man. They are therefore considered as potential targets for melanoma immunotherapy. In this study, we have used the attenuated auxotrophic mutant strain SL7207 of Salmonella typhimurium as vehicle for a human gp100 (hgp100) DNA vaccine against melanoma. In vitro studies indicate that Salmonella/pCMV-hgp100 is efficiently scavenged by dendritic cells, resulting in the expression of the hgp100 transcription unit in the DC. In addition, oral administration of Salmonella/pCMV-hgp100 results in the expression of hgp100 RNA and protein by cells exhibiting DC-morphology in mesenteric lymph nodes as soon as 3 days after vaccination. Analysis of the efficacy of the Salmonella/pCMV-hgp100 vaccine in the B16/hgp100 model demonstrated the induction of strong anti-hgp100 CTL responses and protective immunity in 70% of the vaccinated mice, but not in control mice. Based on these data, we consider S. typhimurium as a useful vehicle for the design of recombinant DNA based anti-cancer vaccines.

The Presence of Small Intestinal Intraepithelial Gamma/Delta T-Lymphocytes Is Inversely Correlated With Lymphoma Development in Refractory Celiac Disease

BACKGROUND:In refractory celiac disease (RCD) type II, a phenotypically aberrant (CD7+ CD3− CD4/8-cytoplasmicCD3+) intraepithelial lymphocyte (IEL) population is present, and 50–60% of these patients develop enteropathy-associated T-cell lymphoma (EATL). TCRγδ+ IELs play an important role in mucosal repair, homeostasis, and tumor surveillance. Recently, human small intestinal TCRγδ+ IELs were shown to have regulatory potential in uncomplicated celiac disease (CD).AIM:In the present study, we investigated whether TCRγδ+ IELs are decreased in RCD II, providing a possible explanation for persisting mucosal damage and inflammation, and the emergence of aberrant T cells with clonal expansion to EATL.DESIGN AND METHODS:Multiparameter flow cytometric immunophenotyping was performed on IELs isolated from fresh small bowel biopsy specimens of relatively large distinct CD patient and control groups (N = 87).RESULTS:A significantly lower percentage of TCRγδ+ IELs was found in RCD II as compared to all other CD groups. In contrast, in uncomplicated CD patients significantly more TCRγδ+ IELs were found than in controls. Overall, there is a clear negative relation between TCRγδ+ IELs and aberrant IELs. Interestingly, TCRγδ+ IELs increase again in RCD II after effective therapy.CONCLUSIONS:The observed negative relation between TCRγδ+ and aberrant IELs, along with their known regulatory capacity in uncomplicated CD, implies that TCRγδ+ IELs may play a crucial role in mucosal repair, regaining homeostasis and possibly even tumor surveillance. These cells may be important markers, in addition to the aberrant T cells, to differentiate between disease categories and to evaluate the effectiveness of therapeutic strategies.

Astrocyte-Derived Tissue Transglutaminase Interacts with Fibronectin: A Role in Astrocyte Adhesion and Migration?

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring.

Maggot excretions affect the human complement system

The complement system plays an important role in the activation of the inflammatory response to injury, although inappropriate complement activation (CA) can lead to severe tissue damage. Maggot therapy is successfully used to treat infected wounds. In this study, we hypothesized that maggot excretions/secretions influence CA in order to modulate the hosts inflammatory response. Therefore, the effect of maggot excretions on CA was investigated in preoperatively and postoperatively obtained sera from patients. Our results show that maggot excretions reduce CA in healthy and postoperatively immune‐activated human sera up to 99.9%, via all pathways. Maggot excretions do not specifically initiate or inhibit CA, but break down complement proteins C3 and C4 in a cation‐independent manner and this effect proves to be temperature tolerant. This study indicates a CA‐reducing substrate that is already successfully used in clinical practice and may explain part of the improved wound healing caused by maggot therapy. Furthermore, the complement activation‐reducing substance present in maggot excretions could provide a novel treatment modality for several diseases, resulting from an (over)active complement system.

Identification of a potential human telomerase reverse transcriptase–derived, HLA-A1–restricted cytotoxic T-lymphocyte epitope

The catalytic subunit of human telomerase reverse transcriptase (hTERT) is expressed in the majority of tumor cells of different histological origins as opposed to most normal somatic cells. This implicates hTERT as a widely expressed tumor-associated antigen and an attractive candidate for antigen-specific tumor immunotherapy. T lymphocytes specific for hTERT-derived epitopes have been isolated and shown reactive with hTERT-expressing tumor cells. To further increase the applicability of hTERT as a target antigen for immunotherapy, we set out to identify potential hTERT-derived, HLA-A1–restricted cytotoxic T-lymphocyte (CTL) epitopes. The “reverse immunology” approach, involving computer-assisted epitope prediction, in vitro CTL induction, and tetramer-guided CTL isolation, resulted in specific CTLs against hTERT-derived, HLA-A1–binding peptides. Intermediate- to low-avidity CTLs were induced against the hTERT325-333 peptide and recognized endogenously processed hTERT. Recognition of endogenous hTERT depended on an increase of hTERT expression above normal levels in tumor cells through hTERT transduction, most probably as a result of limited CTL avidity. The altered peptide ligand hTERT699T-707 was designed to increase HLA-A1–binding affinity of the hTERT699-707 peptide and was used to induce CTLs. However, these CTLs poorly cross-recognized native hTERT699-707 and failed to recognize endogenously processed hTERT. In conclusion, our study has identified the hTERT325-333 peptide as a potential hTERT-derived epitope that may prove useful for induction and monitoring of hTERT-specific, HLA-A1–restricted CTL responses.