Resolution of Acinar Dedifferentiation Regulates Tissue Remodeling in Pancreatic Injury and Cancer Initiation.

BACKGROUND & AIMS: Acinar-to-ductal metaplasia (ADM) is crucial in the development of pancreatic ductal adenocarcinoma. However, our understanding of the induction and resolution of ADM remains limited. We conducted comparative transcriptome analyses to identify conserved mechanisms of ADM in mouse and human. METHODS: We identified Sox4 among the top up-regulated genes. We validated the analysis by RNA in situ hybridization. We performed experiments in mice with acinar-specific deletion of Sox4 (Ptf1a: CreER; Rosa26-LSL-YFPLSL-YFP; Sox4fl/fl) with and without an activating mutation in Kras (KrasLSL-G12D/+). Mice were given caerulein to induce pancreatitis. We performed phenotypic analysis by immunohistochemistry, tissue decellularization, and single-cell RNA sequencing. RESULTS: We demonstrated that Sox4 is reactivated in ADM and pancreatic intraepithelial neoplasias. Contrary to findings in other tissues, Sox4 actually counteracts cellular dedifferentiation and helps maintain tissue homeostasis. Moreover, our investigations unveiled the indispensable role of Sox4 in the specification of mucin-producing cells and tuft-like cells from acinar cells. We identified Sox4-dependent non-cell-autonomous mechanisms regulating the stromal reaction during disease progression. Notably, Sox4-inferred targets are activated upon KRAS inactivation and tumor regression. CONCLUSIONS: Our results indicate that our transcriptome analysis can be used to investigate conserved mechanisms of tissue injury. We demonstrate that Sox4 restrains acinar dedifferentiation and is necessary for the specification of acinar-derived metaplastic cells in pancreatic injury and cancer initiation and is activated upon Kras ablation and tumor regression in mice. By uncovering novel potential strategies to promote tissue homeostasis, our findings offer new avenues for preventing the development of pancreatic ductal adenocarcinoma.

Prognostic importance of margin clearance in ampullary adenocarcinoma following pancreaticoduodenectomy in a Danish population-based nationwide study.

BACKGROUND: In this Danish nationwide population-based study, we evaluated the prognostically relevant minimum tumour-free margin width following pancreaticoduodenectomy (PD) for ampullary adenocarcinoma (AAC) and evaluated whether certain margins hold independent prognostic information. METHODS: We included 128 patients who underwent PD for AAC from 2015 to 2019. Clinical and pathological data including well-known prognostic factors were retrieved from the Danish Pancreatic Cancer Database. Missing data were obtained by review of pathology reports and re-microscopy of resection specimens. All PD specimens were examined using a standardised pathological protocol including multicolour inking, axial slicing and exact reporting of margin widths. The cohort was dichotomised into involved and uninvolved groups, using different margin clearance definitions (0.5-≥3.0 mm). RESULTS: Following PD for AAC, margin clearance of ≥1 mm was independently associated with improved chance of survival compared with <1 mm (HR: 0.30, 95 % CI: 0.14-0.64 (p = 0.002)). Posterior and anterior margin widths were narrower compared with superior mesenteric artery and vein margins. Posterior margin and anterior surface had isolated prognostic significance in multivariable analysis. CONCLUSION: Following PD for AAC, margin clearance of at least 1 mm is independently associated with improved survival. Our data further indicate that anterior surface and posterior margin hold particular prognostic value.

Targeted next-generation sequencing of EUS-guided through-the-needle-biopsy sampling from pancreatic cystic lesions.

BACKGROUND AND AIMS: Recent advances have introduced molecular subtyping of pancreatic cystic lesions (PCLs) as a possible amendment to the diagnostic algorithm. The study evaluated the feasibility and diagnostic accuracy of molecular analysis and subtyping of PCLs using the recently introduced EUS-guided through-the-needle-biopsy (TTNB) sampling. METHODS: We prospectively included 101 patients in the study who presented with PCLs >15 mm in the largest cross-section. EUS-guided TTNB samples were obtained by a micro-biopsy forceps introduced through a 19-gauge needle. The TTNB samples were analyzed by next-generation sequencing (NGS) for point mutations in tumor suppressors and oncogenes using a 51-gene customized hotspot panel. Sensitivity and specificity were calculated with the histologic diagnosis as reference. RESULTS: After initial microscopic evaluation of the samples, 91 patients had residual TTNB samples available for NGS. Of these, 49 harbored mutations, most frequently in KRAS and GNAS, reflecting an excess frequency of intraductal papillary mucinous neoplasms (IPMNs) in the study population. A sensitivity and specificity of 83.7% (95% confidence interval [CI], 70.3-92.7) and 81.8% (95% CI, 48.2-97.7), respectively, were demonstrated for the diagnosis of a mucinous cyst and 87.2% (95% CI, 74.2-95.2) and 84.6% (95% CI, 54.5-98.1) for the diagnosis of an IPMN. CONCLUSIONS: Thus, molecular analysis of TTNB samples by NGS has high sensitivity and specificity for diagnosing mucinous cysts and IPMNs. Although the procedure comes with a risk of adverse events of 9.9%, TTNB samples are a robust alternative to cyst fluid for a combined histologic and molecular diagnosis of PCLs. (Clinical trial registration number: NCT03578445.).

Prognostic significance of margin clearance in pancreaticoduodenectomy specimens with pancreatic ductal adenocarcinoma in a Danish population-based nationwide study.

BACKGROUND: In this nationwide population-based cohort study, we investigated the overall minimum margin width that is independently associated with improved survival following pancreaticoduodenectomy (PD) for pancreatic ductal adenocarcinoma (PDAC) and evaluated whether certain margins or surfaces hold independent prognostic significance. METHODS: Data from 367 patients who underwent PD for PDAC in the period 2015-2019 were retrieved from the Danish Pancreatic Cancer Database. Missing data were obtained by review of pathology reports and re-microscopy of resection specimens. Surgical specimens were evaluated using a standardised pathological protocol involving multicolour inking, axial slicing and exact reporting of circumferential margin clearances in 0.5 mm increments. RESULTS: When categorised according to margin widths of <0.5, <1.0, <1.5, <2.0, <2.5 and <3.0 mm, R1 resections were detected in 34%, 57%, 75%, 78%, 86% and 87% of cases, respectively. In multivariable analyses, an overall margin clearance of ≥1.5 mm was associated with improved survival compared with a clearance of <1.5 mm (HR 0.70 95% CI 0.51-0.97 (p = 0.031)). When evaluating the margins separately, no margin had independent prognostic significance. CONCLUSION: Margin clearance of at least 1.5 mm was independently associated with improved survival following PD for PDAC.

Clinical impact of endoscopic ultrasound-guided through-the-needle microbiopsy in patients with pancreatic cysts.

BACKGROUND: The limited data on the utility of endoscopic ultrasound (EUS)-guided through-the-needle biopsies (TTNBs) in patients with pancreatic cystic lesions (PCLs) originate mainly from retrospective studies. Our aim was to determine the clinical impact of TTNBs, their added diagnostic value, and the adverse event rate in a prospective setting. METHODS: This was a prospective, single-center, open-label controlled study. Between February 2018 and August 2019, consecutive patients presenting with a PCL of 15 mm or more and referred for EUS were included. Primary outcome was a change in clinical management of PCLs following TTNB compared with cross-sectional imaging and cytology. Adverse events were defined according to the ASGE lexicon. RESULTS: 101 patients were included. TTNBs led to a change in clinical management in 11.9 % of cases (n = 12). Of these, 10 had serous cysts and surveillance was discontinued, while one of the remaining two cases underwent surgery following diagnosis of a mucinous cystic neoplasm. The diagnostic yield of TTNBs for a specific cyst diagnosis was higher compared with FNA cytology (69.3 % vs. 20.8 %, respectively; P < 0.001). The adverse event rate was 9.9 % (n = 10; 95 % confidence interval 5.4 % - 17.3 %), with the most common event being acute pancreatitis (n = 9). Four of the observed adverse events were severe, including one fatal outcome. CONCLUSIONS: TTNBs resulted in a change of clinical management in about one in every 10 patients; however, the associated adverse event risk was substantial. Further studies are warranted to elucidate in which subgroups of patients the clinical benefit outweighs the risks.

Clinical value of serum hyaluronan and propeptide of type III collagen in patients with pancreatic cancer.

Hyaluronan (HA) and collagen are highly expressed in pancreatic cancer (PC) stroma. HA and collagen accumulation increase tumor interstitial fluid pressure, compromising blood flow and drug penetration. The aim of this biomarker study was to determine the clinical utility of serum HA and the propeptide of type III collagen (PRO-C3) in patients with PC. A cohort from the Danish BIOPAC study (NCT03311776) including patients with histologically confirmed pancreatic ductal adenocarcinoma (n = 809), ampullary carcinoma (n = 44), distal biliary tract cancer (n = 31), chronic pancreatitis (n = 15), intraductal papillary mucinous neoplasm (n = 41), duodenal adenoma (n = 7) and no cancer (n = 25). Healthy controls were available for serum HA (n = 141) and PRO-C3 (n = 8). The main outcome was overall survival (OS) of patients with PC in relation to pretreatment serum HA and PRO-C3 levels. Patients with PC had higher baseline serum HA and PRO-C3 than healthy subjects and patients with benign conditions. Pretreatment serum baseline HA and PRO-C3 in patients with PC were associated with poorer survival and PRO-C3 remained prognostic also after adjusting for age, performance status, stage, the presence of liver and peritoneum metastasis, and CA19-9. Detection of HA and PRO-C3 may be useful in differentiating between malignant and benign pancreatic conditions. Serum HA and PRO-C3 were prognostic for OS in patients with PC.

Prognostic impact of Charlson's Age-Comorbidity Index and other risk factors in patients with pancreatic cancer.

OBJECTIVES: Few studies have evaluated the impact of risk factors and comorbidity on overall survival (OS) in patients with pancreatic ductal adenocarcinoma (PDAC). The aim was to investigate the prognostic importance of Charlson's age-comorbidity index (CACI) and other risk factors on prognosis in a clinical real-world cohort of PDAC patients. METHODS: A total of 1,159 patients with PDAC who had received at least one cycle of adjuvant or palliative chemotherapy were included from the Danish BIOPAC study. We analysed OS according to CACI, tobacco smoking, alcohol intake, performance status (PS), BMI and diabetes. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated for OS using Cox proportional hazards regression. RESULTS: At the end of follow-up, 994 (86%) patients had died. The median OS was 298 days for all patients (range 3-3010) and shortest in patients with stage IV. No association with short OS was seen for CACI > 2, diabetes, alcohol abuse, tobacco smoking, hypertension, and high BMI. Multivariate analysis showed that stage (IV vs. I: HR = 9.05, 95% CI 5.17-15.84), PS (2 vs. 0: HR = 3.67, 2.92-4.61) and treatment with angiotensin-converting enzyme inhibitors (yes vs. no: HR = 1.31, 1.06-1.61) were independent negative prognostic factors. CONCLUSIONS: We found that CACI, diabetes, tobacco smoking, alcohol abuse, hypertension, and high BMI were not associated with OS in a real-world cohort of patients with PDAC treated with chemotherapy. Only stage and PS were prognostic parameters.

A rare missense variant in APC interrupts splicing and causes AFAP in two Danish families.

BACKGROUND: We report the first case of a missense variant in the APC gene that interrupts splicing by creating a new cryptic acceptor site. The variant, c.289G>A, p.(Gly97Arg), is located in exon 3, and qualitative and semi-quantitative RNA splicing analysis reveal that the variant results in skipping of the last 70 nucleotides of the exon, which leads to the introduction of a frameshift and a premature stop codon. CASE PRESENTATION: The variant was detected in two, apparently unrelated, Danish families with an accumulation of colorectal cancers, colonic adenomas and other cancers. The families both have an attenuated familial adenomatous polyposis phenotype, which is consistent with the association of pathogenic variants in the 5' end of the gene.One variant-carrier also had Caroli Disease and a Caroli Disease associated hepatic mucinous cystadenocarcinoma. This is the first description of a person with both Caroli Disease and a pathogenic APC variant, and although the APC variant is not known to be connected to the development of the hepatic malformations in Caroli Disease, it remains unclear whether the variant could have contributed to the carcinogenesis of the liver tumour. CONCLUSIONS: Based on functional and co-segregation data we classify the APC c.289G>A, p.(Gly97Arg) variant as pathogenic (class 5). Our findings emphasize the importance of a functional evaluation of missense variants although located far from the exon-intron boundaries.

Fenofibrate increases very-long-chain sphingolipids and improves blood glucose homeostasis in NOD mice.

AIMS/HYPOTHESIS: Sphingolipid metabolism regulates beta cell biology and inflammation and is abnormal at the onset of type 1 diabetes. Fenofibrate, a regulator of sphingolipid metabolism, is known to prevent diabetes in NOD mice. Here, we aimed to investigate the effects of fenofibrate on the pancreatic lipidome, pancreas morphology, pancreatic sympathetic nerves and blood glucose homeostasis in NOD mice. METHODS: We treated female NOD mice with fenofibrate from 3 weeks of age. The pancreatic lipidome was analysed using MS. Analysis of pancreas and islet volume was performed by stereology. Islet sympathetic nerve fibre volume was evaluated using tyrosine hydroxylase staining. The effect on blood glucose homeostasis was assessed by measuring non-fasting blood glucose from age 12 to 30 weeks. Furthermore, we measured glucose tolerance, fasting insulin and glucagon levels, and insulin tolerance. RESULTS: We found that fenofibrate selectively increases the amount of very-long-chain sphingolipids in the pancreas of NOD mice. In addition, we found that fenofibrate causes a remodelling of the pancreatic lipidome with an increased amount of lysoglycerophospholipids. Fenofibrate did not affect islet or pancreas volume, but led to a higher volume of islet sympathetic nerve fibres and tyrosine hydroxylase-positive cells. Fenofibrate-treated NOD mice had a more stable blood glucose, which was associated with reduced non-fasting and increased fasting blood glucose. Furthermore, fenofibrate improved glucose tolerance, reduced fasting glucagon levels and prevented fasting hyperinsulinaemia. CONCLUSIONS/INTERPRETATION: These data indicate that fenofibrate alters the pancreatic lipidome to a more anti-inflammatory and anti-apoptotic state. The beneficial effects on islet sympathetic nerve fibres and blood glucose homeostasis indicate that fenofibrate could be used as a therapeutic approach to improve blood glucose homeostasis and prevent diabetes-associated pathologies.

Next-generation sequencing of endoscopic ultrasound guided microbiopsies from pancreatic cystic neoplasms.

AIMS: Interpretation of cytology samples from pancreatic cysts is challenging. A novel microbiopsy forceps used during endoscopic ultrasound examinations offers new opportunities for histological examination of tissue from pancreatic cysts as well as next-generation sequencing. The aim of this study was to analyse the results of next-generation sequencing of microbiopsies from pancreatic cysts. METHODS AND RESULTS: Microbiopsies from 27 patients were obtained, 23 of which were subjected to next-generation sequencing. Sixteen intraductal papillary mucinous neoplasms harboured mutations in genes regulating cell cycle and repair, and three were without mutations. Most frequent mutations were found in the KRAS and GNAS genes, and these were often concomitant. Three serous cystic neoplasms were without mutations, while with regard to histology, a non-diagnostic microbiopsy harboured a KRAS and a TP53 mutation and was deemed malignant after clinical follow-up. Three patients underwent surgery, and the point mutations detected in the microbiopsies were confirmed in the resected specimens. We identified one resected sample with an additional GNAS mutation which was not identified in the microbiopsy. CONCLUSIONS: Next-generation sequencing of microbiopsies may have the potential to improve diagnostic decision-making.

Abnormal islet sphingolipid metabolism in type 1 diabetes.

AIMS/HYPOTHESIS: Sphingolipids play important roles in beta cell physiology, by regulating proinsulin folding and insulin secretion and in controlling apoptosis, as studied in animal models and cell cultures. Here we investigate whether sphingolipid metabolism may contribute to the pathogenesis of human type 1 diabetes and whether increasing the levels of the sphingolipid sulfatide would prevent models of diabetes in NOD mice. METHODS: We examined the amount and distribution of sulfatide in human pancreatic islets by immunohistochemistry, immunofluorescence and electron microscopy. Transcriptional analysis was used to evaluate expression of sphingolipid-related genes in isolated human islets. Genome-wide association studies (GWAS) and a T cell proliferation assay were used to identify type 1 diabetes related polymorphisms and test how these affect cellular islet autoimmunity. Finally, we treated NOD mice with fenofibrate, a known activator of sulfatide biosynthesis, to evaluate the effect on experimental autoimmune diabetes development. RESULTS: We found reduced amounts of sulfatide, 23% of the levels in control participants, in pancreatic islets of individuals with newly diagnosed type 1 diabetes, which were associated with reduced expression of enzymes involved in sphingolipid metabolism. Next, we discovered eight gene polymorphisms (ORMDL3, SPHK2, B4GALNT1, SLC1A5, GALC, PPARD, PPARG and B4GALT1) involved in sphingolipid metabolism that contribute to the genetic predisposition to type 1 diabetes. These gene polymorphisms correlated with the degree of cellular islet autoimmunity in a cohort of individuals with type 1 diabetes. Finally, using fenofibrate, which activates sulfatide biosynthesis, we completely prevented diabetes in NOD mice and even reversed the disease in half of otherwise diabetic animals. CONCLUSIONS/INTERPRETATION: These results indicate that islet sphingolipid metabolism is abnormal in type 1 diabetes and suggest that modulation may represent a novel therapeutic approach. DATA AVAILABILITY: The RNA expression data is available online at https://www.dropbox.com/s/93mk5tzl5fdyo6b/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%2C%20RNA%20expression.xlsx?dl=0 . A list of SNPs identified is available at https://www.dropbox.com/s/yfojma9xanpp2ju/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%20SNP.xlsx?dl=0 .

A novel endoscopic ultrasound-guided through-the-needle microbiopsy procedure improves diagnosis of pancreatic cystic lesions.

BACKGROUND: Pancreatic cystic lesions represent a diagnostic dilemma as some may harbor malignancy or have potential for malignant transformation. The aim of this study was to present our experience with a novel endoscopic ultrasound (EUS)-guided microbiopsy procedure enabling procurement of tissue from the wall of the cystic lesion. METHODS: We collected data from 31 consecutive patients with pancreatic cystic lesions who underwent an EUS-guided microbiopsy procedure at our institution. Records were retrospectively reviewed from a prospectively maintained database. RESULTS: The technical success was 87.1 %. Diagnostic yield of microbiopsies was 71.0 %. Microbiopsies offered sufficient tissue for morphological and immunohistochemical characterization of the lesions, as well as determination of grade of dysplasia. Furthermore, evaluation of microbiopsies changed the clinical management in six patients (19.4 %). Three nonsevere adverse events were observed (9.7 %): two cases of mild infection and one case of mild pancreatitis. All three patients recovered completely. CONCLUSIONS: EUS-guided microbiopsy procedure was technically feasible, with a high diagnostic yield. Further prospective studies are needed to confirm these promising results.

Quantitative perfusion assessment of intestinal anastomoses in pigs treated with glucagon-like peptide 2.

PURPOSE: Despite exhaustive research and improvement of techniques, anastomotic leakage remains a frequent complication in gastrointestinal surgery. As leakage is associated with poor perfusion, reliable objective methods to assess anastomotic perfusion are highly demanded. In addition, such methods enable evaluation of interventions that may improve anastomotic perfusion. Glucagon-like peptide 2 (GLP-2) is an enteroendocrine hormone that regulates mid-gut perfusion. In the present study, we aimed to explore if quantitative perfusion assessment with indocyanine green (q-ICG) could detect an increase in porcine anastomotic perfusion after treatment with GLP-2. METHODS: Nineteen pigs had two small bowel resections followed by anastomosis. Blinded to all investigators, animals were randomized to receive GLP-2 or placebo. Anastomotic perfusion was assessed at baseline, 30 min after injection of GLP-2/placebo, and after 5 days of treatment. Anastomotic strength and healing were evaluated by bursting pressure and histology. RESULTS: Q-ICG detected a significantly higher increase in anastomotic perfusion (p < 0.05) in animals treated with GLP-2, compared with placebo. No significant differences in anastomotic strength or healing were found. CONCLUSIONS: Q-ICG is a promising tool for perfusion assessment in gastrointestinal surgery and opens new opportunities in research of factors that may influence anastomotic healing, but further research is warranted to evaluate the effects of GLP-2 on anastomotic healing.

Immunohistochemical assessment of glucagon-like peptide 1 receptor (GLP-1R) expression in the pancreas of patients with type 2 diabetes.

AIMS: Glucagon-like peptide-1 (GLP-1) is an incretin hormone which stimulates insulin release and inhibits glucagon secretion from the pancreas in a glucose-dependent manner. Incretin-based therapies, consisting of GLP-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, are used for the treatment of type 2 diabetes (T2D). Immunohistochemical studies for GLP-1R expression have been hampered previously by the use of unspecific polyclonal antibodies. This study aimed to assess the expression levels of GLP-1R in a set of T2D donor samples obtained via nPOD. METHODS: This study used a new monoclonal antibody to assess GLP-1R expression in pancreatic tissue from 23 patients with T2D, including 7 with a DPP-4 inhibitor and 1 with a history of GLP-1R agonist treatment. A software-based automated image analysis algorithm was used for quantitating intensities and area fractions of GLP-1R positive compartments. RESULTS: The highest intensity GLP-1R immunostaining was seen in beta-cells in islets (average signal intensity, 76.1 [±8.1]). GLP-1R/insulin double-labelled single cells or small clusters of cells were also frequently located within or in close vicinity of ductal epithelium in all samples and with the same GLP-1R immunostaining intensity as found in beta-cells in islets. In the exocrine pancreas a large proportion of acinar cells expressed GLP-1R with a 3-fold lower intensity of immunoreactivity as compared to beta-cells (average signal intensity 25.5 [±3,3]). Our studies did not unequivocally demonstrate GLP-1R immunoreactivity on normal-appearing ductal epithelium. Pancreatic intraepithelial neoplasia (PanINs; a form of non-invasive pancreatic ductular neoplasia) was seen in most samples, and a minority of these expressed low levels of GLP-1R. CONCLUSION: These data confirm the ubiquity of early stage PanIN lesions in patients with T2D and do not support the hypothesis that incretin-based therapies are associated with progression towards the more advanced stage PanIN lesions.

Molecular biomarkers have the potential to improve the diagnostic work-up of pancreatic cystic lesions.

Pancreatic cysts are increasingly diagnosed due to the widespread use of cross-sectional imaging, and some of these lesions harbor malignant potential. Mucinous cystic neoplasms and intraductal papillary mucinous neoplasms are the major premalignant cystic neoplasms of the pancreas. A variety of diagnostic tools are used to predict the malignant potential of these cysts, but specificity and sensitivity are limited. Thus, many patients undergo unnecessary operations for benign cysts. Balancing the risks of watchful waiting with those of operative management is key in managing these lesions. During the last decade, genetic changes of pancreatic cysts have been examined extensively to estimate their malignant potential. In this review, we provide an overview of the latest molecular and genetic aspects of pancreatic cysts and how they may contribute to the differential diagnosis in patients with pancreatic cystic neoplasms.

Mode of delivery shapes gut colonization pattern and modulates regulatory immunity in mice.

Delivery mode has been associated with long-term changes in gut microbiota composition and more recently also with changes in the immune system. This has further been suggested to link Cesarean section (C-section) with an increased risk for development of immune-mediated diseases such as type 1 diabetes. In this study, we demonstrate that both C-section and cross-fostering with a genetically distinct strain influence the gut microbiota composition and immune key markers in mice. Gut microbiota profiling by denaturing gradient gel electrophoresis and 454/FLX-based 16S rRNA gene amplicon sequencing revealed that mice born by C-section had a distinct bacterial profile at weaning characterized by higher abundance of Bacteroides and Lachnospiraceae, and less Rikenellaceae and Ruminococcus. No clustering according to delivery method as determined by principal component analysis of denaturing gradient gel electrophoresis profiles was evident in adult mice. However, the adult C-section-born mice had lower proportions of Foxp3(+) regulatory T cells, tolerogenic CD103(+) dendritic cells, and less Il10 gene expression in mesenteric lymph nodes and spleens. This demonstrates long-term systemic effect on the regulatory immune system that was also evident in NOD mice, a model of type 1 diabetes, born by C-section. However, no effect of delivery mode was seen on diabetes incidence or insulitis development. In conclusion, the first exposure to microorganisms seems to be crucial for the early life gut microbiota and priming of regulatory immune system in mice, and mode of delivery strongly influences this.