Targeted delivery of antisense oligonucleotides to pancreatic ß-cells.

Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting ß-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic ß-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.

Differential expression of chemokine receptors on human IgA+ and IgG+ B cells.

Organ-specific lymphocyte homing is dependent on the expression of tissue-specific homing receptors and selected chemokine receptors. During the effector phase of an immune response, IgA and IgG antibody-secreting cells (ASC) are differently distributed in the body. Still, B cell expression of L-selectin and the mucosal homing receptor integrin alpha4beta7 is not related to the isotype produced, but only to the site of antigen encounter. In this study, we examined if differences in chemokine responsiveness between IgA+ and IgG+ B cells could explain their different tissue localization. Circulating CD19+ B cells were isolated and their expression of IgA, IgG, and selected chemokine receptors was determined by flow cytometry. Few Ig+ cells expressed CCR2, CCR3, or CCR9, and there was no difference in the expression of these receptors between IgA+ and IgG+ cells. In contrast, CCR4, CCR5, and CXCR3 was expressed on significantly more IgG+ than IgA+ cells. The function of chemokine receptors on memory B cells and ASC was then tested in the transwell system. IgG+ memory cells migrated to a higher extent than IgA+ cells towards the CXCR3 ligand CXCL11/I-TAC, while there was only a small migration towards the CCR4 ligand CCL17/TARC and the CCR9 ligand CCL25/TECK. ASC migrated poorly to all chemokines tested. In conclusion, this study shows that IgG+ and IgA+ memory B cells have a differential expression of the Th1 associated chemokine receptor CXCR3, as well as of CCR4 and CCR5. In contrast, none of the studied chemokine receptors was preferentially expressed by IgA+ cells.

Human gastric B cell responses can be induced by intestinal immunisation.

BACKGROUND: In a previous study, we found that oral vaccination induces strong B cell responses in the stomach of Helicobacter pylori infected but not of uninfected individuals. In this study, we have evaluated the possibility of inducing gastric immune responses in H pylori infected volunteers by intestinal and gastric immunisation. METHODS: H pylori infected subjects were given two doses of an inactivated cholera vaccine, either intestinally via an endoscope approximately 30 cm distal to the pylorus sphincter or intragastrically as small droplets applied directly onto the stomach mucosa. Uninfected individuals received the vaccine by standard oral procedure. Vaccine specific antibody secreting cells in antral and duodenal biopsies were detected by the enzyme linked immunospot assay technique before and seven days after the second immunisation. RESULTS: Intestinal immunisations resulted in induction of vaccine specific gastric IgA secreting cells in five of eight volunteers. This immunisation schedule also gave rise to specific duodenal antibody secreting cells in seven of eight individuals. Local gastric immunisation resulted in the induction of specific B cells in the gastric mucosa of four of eight volunteers. Gastric antigen application also resulted in B cell responses in the duodenum in all volunteers. Uninfected volunteers receiving the vaccine perorally responded in the duodenum but not in the stomach. CONCLUSIONS: H pylori infection increases the ability of the gastric mucosa to serve as an expression site for intestinally induced B cell responses. These findings are of importance when designing a therapeutic H pylori vaccine, and based on our results such a vaccine can be delivered along the whole upper gastrointestinal tract.

Homing commitment of lymphocytes activated in the human gastric and intestinal mucosa.

BACKGROUND: Gastric infection with the human pathogen Helicobacter pylori results in a large accumulation of IgA and IgM secreting cells in the gastric mucosa. The molecular mechanisms resulting in B cell migration to the gastric mucosa in H pylori infection are however not known. AIMS: To examine expression of the mucosal homing receptor integrin alpha4beta7 and the homing receptor for secondary lymphoid tissues, L-selectin, on lymphocytes activated by gastric, intestinal, or systemic antigens. Furthermore, to examine gastric expression of the mucosal addressin cellular adhesion molecule 1 (MAdCAM-1), the endothelial counter-receptor to integrin alpha4beta7. SUBJECTS AND METHODS: H pylori infected individuals were immunised by either gastric (n=8) or intestinal (n=8) delivery of an inactivated cholera vaccine. The resulting circulating vaccine specific B cells were sorted according to alpha4beta7 and L-selectin expression and assayed for production of IgA and IgG using an enzyme linked immunospot assay. In addition, circulating CD4+ T cells from seven H pylori infected individuals were fractionated according to alpha4beta7 and L-selectin expression. The resulting T cell fractions were then assayed for specific proliferation against H pylori or the systemic antigen tetanus toxoid. Finally, gastric expression of MAdCAM-1 was determined by immunohistochemistry in H pylori infected (n=16) and uninfected (n=8) individuals. RESULTS: Virtually all B cells induced by both gastric and intestinal antigen delivery expressed alpha4beta7 whereas less then half coexpressed L-selectin. Furthermore, H pylori reactive T cells were mainly found in the alpha4beta7+L-selectin+ T cell fraction whereas tetanus specific T cells were largely alpha4beta7-L-selectin+. MAdCAM-1 was present in similar amounts in gastric mucosa from H pylori infected and uninfected individuals. CONCLUSIONS: B cells and T cells activated by antigens delivered to the gastric mucosa express the mucosal homing receptor integrin alpha4beta7, as do cells activated in the intestine. Together with the observation that gastric endothelial cells express MAdCAM-1, this may partly explain the homing of lymphocytes activated in the stomach or in the small intestine to the gastric mucosa.

Role of local cytokines in increased gastric expression of the secretory component in Helicobacter pylori infection.

Using immunohistochemical staining, we examined the presence of secretory component (SC) on epithelial cells in gastric and duodenal biopsy specimens collected from Helicobacter pylori-infected individuals and healthy controls. Gastric epithelial cells from healthy volunteers expressed low, but detectable, levels of SC. In contrast, significantly higher level of expression of SC (P < 0.001) was observed on epithelial cells in the antra of H. pylori-infected individuals. The antral SC expression correlated with staining for gamma interferon of intraepithelial and lamina propria lymphocytes (r(s) = 0.76 and 0.69, respectively, P < 0.001) and correlated weakly with production of tumor necrosis factor alpha (r(s) = 0.43, P < 0.05), but it did not correlate at all with interleukin-4 production.

Antibody-secreting cells in the stomachs of symptomatic and asymptomatic Helicobacter pylori-infected subjects.

In this study we analyzed whether infection with Helicobacter pylori gives rise to specific B-cell responses against a number of putative virulence factors of H. pylori, e.g., urease, flagellin, and different bacterial surface antigens, locally in the gastric mucosa. This was studied in antrum and corpus biopsies collected from 11 H. pylori-infected patients with duodenal ulcers, 11 asymptomatic H. pylori carriers, and 13 noninfected, healthy controls. Mononuclear cells were isolated from the biopsies and assayed for frequencies of total and H. pylori-specific antibody-secreting cells (ASCs) by means of the enzyme-linked immunospot technique. The H. pylori-infected subjects had remarkably higher frequencies of total immunoglobulin A (IgA)- and IgM-secreting cells than the noninfected subjects, while the frequencies of IgG-secreting cells were virtually the same in the different groups. In addition, most of the infected subjects had IgA ASCs reacting with H. pylori membrane proteins, flagellin, and urease, while none of the noninfected subjects had any detectable H. pylori-reactive ASCs. Furthermore, half of the infected subjects also had ASCs reacting with a Helicobacter-specific 26-kDa protein, while only a few of them had ASCs reacting with neutrophil-activating protein, the neuraminyllactose-binding hemagglutinin HpaA, or lipopolysaccharides purified from different H. pylori strains. The frequencies of H. pylori-specific ASCs in the antrum and corpus were almost identical, and no differences in either antigen specificity or magnitude of the B-cell response in the stomach could be detected between the ulcer patients and the asymptomatic H. pylori carriers. This study demonstrates that H. pylori infection induces strong antibody responses in the human gastric mucosa, both in asymptomatic carriers and in duodenal ulcer patients. However, the outcome of infection could not be explained by differences in the local B-cell response to any of the antigens used in this study.