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The fatal transmissions of highly pathogenic avian influenza A viruses (IAV) of the H5N1 subtype to humans and high titer replication in the respiratory tract indicate that these pathogens can overcome the bird-to-human species barrier. While type I interferons (IFN-α/β) are well described to contribute to the species barrier of many zoonotic viruses, current data to the role of these antiviral cytokines during human H5N1 IAV infections is limited and contradictory. We hypothesized an important role for the IFN system in limiting productive infection of avian H5N1 strains in human cells. Hence, we examined IFN-α/β gene activation by different avian and human H5N1 isolates, if the IFN-α/β response restricts H5N1 growth and whether the different strains were equally capable to regulate the IFN-α/β system via their IFN-antagonistic NS1 proteins. Two human H5N1 isolates and a seasonal H3N2 strain propagated efficiently in human respiratory cells and induced little IFN-β, whereas three purely avian H5N1 strains were attenuated for replication and provoked higher IFN secretion. Replication of avian viruses was significantly enhanced on interferon-deficient cells, and exogenous IFN potently limited the growth of all strains in human cells. Moreover, IFN-α/β activation by all strains depended on retinoic acid-inducible gene I excluding principal differences in receptor activation between the different viruses. Interestingly, all H5N1 NS1 proteins suppressed IFN-α/β induction comparably well to the NS1 of seasonal IAV. Thus, our study shows that H5N1 strains are heterogeneous in their capacity to activate human cells in an NS1-independent manner. Our findings also suggest that H5N1 viruses need to acquire adaptive changes to circumvent strong IFN-α/β activation in human host cells. Since no single amino acid polymorphism could be associated with a respective high- or low induction phenotype we propose that the necessary adaptations to overcome the human IFN-α/β barrier involve mutations in multiple H5N1 genes.

The calicivirus Rabbit haemorrhagic disease virus (RHDV) is widely used in Australia as a biocontrol agent to manage wild European rabbit (Oryctolagus cuniculus) populations. However, widespread herd immunity limits the effectiveness of the currently used strain, CAPM V-351. To overcome this, we developed an experimental platform for the selection and characterisation of novel RHDV strains. As RHDV does not replicate in cell culture, variant viruses were selected by serially passaging a highly virulent RHDV field isolate in immunologically naïve laboratory rabbits that were passively immunised 18-24 hours post-challenge with a neutralising monoclonal antibody. After seven passages, two amino acid substitutions in the P2 domain of the capsid protein became fixed within the virus population. Furthermore, a synonymous substitution within the coding sequence of the viral polymerase appeared and was also maintained in all subsequent passages. These findings demonstrate proof-of-concept that RHDV evolution can be experimentally manipulated to select for virus variants with altered phenotypes, in this case partial immune escape.

The fatal transmissions of highly pathogenic avian influenza A viruses (IAV) of the H5N1 subtype to humans and high titer replication in the respiratory tract indicate that these pathogens can overcome the bird-to-human species barrier. While type I interferons (IFN-[alpha]/[beta]) are well described to contribute to the species barrier of many zoonotic viruses, current data to the role of these antiviral cytokines during human H5N1 IAV infections is limited and contradictory. We hypothesized an important role for the IFN system in limiting productive infection of avian H5N1 strains in human cells. Hence, we examined IFN-[alpha]/[beta] gene activation by different avian and human H5N1 isolates, if the IFN-[alpha]/[beta] response restricts H5N1 growth and whether the different strains were equally capable to regulate the IFN-[alpha]/[beta] system via their IFN-antagonistic NS1 proteins. Two human H5N1 isolates and a seasonal H3N2 strain propagated efficiently in human respiratory cells and induced little IFN-[beta], whereas three purely avian H5N1 strains were attenuated for replication and provoked higher IFN secretion. Replication of avian viruses was significantly enhanced on interferon-deficient cells, and exogenous IFN potently limited the growth of all strains in human cells. Moreover, IFN-[alpha]/[beta] activation by all strains depended on retinoic acid-inducible gene I excluding principal differences in receptor activation between the different viruses. Interestingly, all H5N1 NS1 proteins suppressed IFN-[alpha]/[beta] induction comparably well to the NS1 of seasonal IAV. Thus, our study shows that H5N1 strains are heterogeneous in their capacity to activate human cells in an NS1-independent manner. Our findings also suggest that H5N1 viruses need to acquire adaptive changes to circumvent strong IFN-[alpha]/[beta] activation in human host cells. Since no single amino acid polymorphism could be associated with a respective high- or low induction phenotype we propose that the necessary adaptations to overcome the human IFN-[alpha]/[beta] barrier involve mutations in multiple H5N1 genes.

BACKGROUND: Only one strain (the Czech CAPM-v351) of rabbit haemorrhagic disease virus (RHDV) has been released in Australia and New Zealand to control pest populations of the European rabbit O. cuniculus. Antigenic variants of RHDV known as RHDVa strains are reportedly replacing RHDV strains in other parts of the world, and Australia is currently investigating the usefulness of RHDVa to complement rabbit biocontrol efforts in Australia and New Zealand. RHDV efficiently kills adult rabbits but not rabbit kittens, which are more resistant to RHD the younger they are and which may carry the virus without signs of disease for prolonged periods. These different infection patterns in young rabbits may significantly influence RHDV epidemiology in the field and hence attempts to control rabbit numbers. METHODS: We quantified RHDV replication and shedding in 4–5 week old rabbits using quantitative real time PCR to assess their potential to shape RHDV epidemiology by shedding and transmitting virus. We further compared RHDV-v351 with an antigenic variant strain of RHDVa in kittens that is currently being considered as a potential RHDV strain for future release to improve rabbit biocontrol in Australia. RESULTS: Kittens were susceptible to infection with virus doses as low as 10 ID₅₀. Virus growth, shedding and transmission after RHDVa infection was found to be comparable or non-significantly lower compared to RHDV. Virus replication and shedding was observed in all kittens infected, but was low in comparison to adult rabbits. Both viruses were shed and transmitted to bystander rabbits. While blood titres indicated that 4–5 week old kittens mostly clear the infection even in the absence of maternal antibodies, virus titres in liver, spleen and mesenteric lymph node were still high on day 5 post infection. CONCLUSIONS: Rabbit kittens are susceptible to infection with very low doses of RHDV, and can transmit virus before they seroconvert. They may therefore play an important role in RHDV field epidemiology, in particular for virus transmission within social groups during virus outbreaks.

Graft-derived cell-free DNA (GcfDNA), which is released into the blood stream by necrotic and apoptotic cells, is a promising noninvasive organ integrity biomarker. In liver transplantation (LTx), neither conventional liver function tests (LTFs) nor immunosuppressive drug monitoring are very effective for rejection monitoring. We therefore hypothesized that the quantitative measurement of donor-derived cell-free DNA (cfDNA) would have independent value for the assessment of graft integrity, including damage from acute rejection. Traditional LFTs were performed and plasma GcfDNA was monitored in 115 adults post-LTx at three German transplant centers as part of a prospective, observational, multicenter cohort trial. GcfDNA percentage (graft cfDNA/total cfDNA) was measured using droplet digital PCR (ddPCR), based on a limited number of predefined single nucleotide polymorphisms, enabling same-day turn-around. The same method was used to quantify blood microchimerism. GcfDNA was increased >50% on day 1 post-LTx, presumably from ischemia/reperfusion damage, but rapidly declined in patients without graft injury within 7 to 10 d to a median <10%, where it remained for the 1-y observation period. Of 115 patients, 107 provided samples that met preestablished criteria. In 31 samples taken from 17 patients during biopsy-proven acute rejection episodes, the percentage of GcfDNA was elevated substantially (median 29.6%, 95% CI 23.6%-41.0%) compared with that in 282 samples from 88 patients during stable periods (median 3.3%, 95% CI 2.9%-3.7%; p < 0.001). Only slightly higher values (median 5.9%, 95% CI 4.4%-10.3%) were found in 68 samples from 17 hepatitis C virus (HCV)-positive, rejection-free patients. LFTs had low overall correlations (r = 0.28-0.62) with GcfDNA and showed greater overlap between patient subgroups, especially between acute rejection and HCV+ patients. Multivariable logistic regression modeling demonstrated that GcfDNA provided additional LFT-independent information on graft integrity. Diagnostic sensitivity and specificity were 90.3% (95% CI 74.2%-98.0%) and 92.9% (95% CI 89.3%-95.6%), respectively, for GcfDNA at a threshold value of 10%. The area under the receiver operator characteristic curve was higher for GcfDNA (97.1%, 95% CI 93.4%-100%) than for same-day conventional LFTs (AST: 95.7%; ALT: 95.2%; γ-GT: 94.5%; bilirubin: 82.6%). An evaluation of microchimerism revealed that the maximum donor DNA in circulating white blood cells was only 0.068%. GcfDNA percentage can be influenced by major changes in host cfDNA (e.g., due to leukopenia or leukocytosis). One limitation of our study is that exact time-matched GcfDNA and LFT samples were not available for all patient visits. In this study, determination of GcfDNA in plasma by ddPCR allowed for earlier and more sensitive discrimination of acute rejection in LTx patients as compared with conventional LFTs. Potential blood microchimerism was quantitatively low and had no significant influence on GcfDNA value. Further research, which should ideally include protocol biopsies, will be needed to establish the practical value of GcfDNA measurements in the management of LTx patients.

The role of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in immune responses mediated by T-helper 2 (T H 2) lymphocytes is unknown. Here we characterize the development of allergic airway disease in TRAIL-deficient ( Tnfsf10 −/− ) mice and in mice exposed to short interfering RNA targeting TRAIL. We show that TRAIL is abundantly expressed in the airway epithelium of allergic mice and that inhibition of signaling impairs production of the chemokine CCL20 and homing of myeloid dendritic cells and T cells expressing CCR6 and CD4 to the airways. Attenuated homing limits T H 2 cytokine release, inflammation, airway hyperreactivity and expression of the transcriptional activator STAT6. Activation of STAT6 by interleukin-13 restores airway hyperreactivity in Tnfsf10 −/− mice. Recombinant TRAIL induces pathognomic features of asthma and stimulates the production of CCL20 in primary human bronchial epithelium cells. TRAIL is also increased in sputum of asthmatics. The function of TRAIL in the airway epithelium identifies this molecule as a target for the treatment of asthma.

Abstract Introduction: Joint linkage and association (JLA) analysis combines two disease gene mapping strategies: linkage information contained in families and association information contained in populations. Such a JLA analysis can increase mapping power, especially when the evidence for both linkage and association is low to moderate. Similarly, an association analysis based on haplotypes instead of single markers can increase mapping power when the association pattern is complex. Methods: In this paper, we present an extension to the GENEHUNTER-MODSCORE software package that enables a JLA analysis based on haplotypes and uses information from arbitrary pedigree types and unrelated individuals. Our new JLA method is an extension of the MOD score approach for linkage analysis, which allows the estimation of trait-model and linkage disequilibrium (LD) parameters, i.e., penetrance, disease-allele frequency, and haplotype frequencies. LD is modeled between alleles at a single diallelic disease locus and up to three diallelic test markers. Linkage information is contributed by additional multi-allelic flanking markers. We investigated the statistical properties of our JLA implementation using extensive simulations, and we compared our approach to another commonly used single-marker JLA test. To demonstrate the applicability of our new method in practice, we analyzed pedigree data from the German National Case Collection for Familial Pancreatic Cancer (FaPaCa). Results: Based on the simulated data, we demonstrated the validity of our JLA-MOD score analysis implementation and identified scenarios in which haplotype-based tests outperformed the single-marker test. The estimated trait-model and LD parameters were in good accordance with the simulated values. Our method outperformed another commonly used JLA single-marker test when the LD pattern was complex. The exploratory analysis of the FaPaCa families led to the identification of a promising genetic region on chromosome 22q13.33, which can serve as a starting point for future mutation analysis and molecular research in pancreatic cancer. Conclusion: Our newly proposed JLA-MOD score method proves to be a valuable gene mapping and characterization tool, especially when either linkage or association information alone provide insufficient power to identify the disease-causing genetic variants.

Objective. Neuroendocrine neoplasias (NENs) represent a rare and biologically heterogeneous group of malignancies. Treatment of NEN patients remains challenging due to lack of prospective evidence on the choice of ideal therapeutic sequence and therapeutic efficacy in specific individual scenarios. Methods. Clinical data on 110 consecutive patients suffering from NEN treated at a single German university center were analyzed, therapeutic regimens applied were assessed, and the outcome was evaluated. Results. Histological grading, Ki67 proliferation index, functional activity, and presence of metastases were identified as prognostic markers. 10-year overall survival rates were 92%, 44%, and 0% for G1, G2, and G3 tumors, and 60%, 39%, 69%, 53%, and 0% for Ki67 <2%, 3–5%, 6–20%, 21–49%, and >50%, respectively. Peptide receptor radionuclide therapy (PRRT) and cytostatic chemotherapy were the second most common options, with PRRT being used more frequently in NET G1 and G2 and chemotherapy in NEC G3. Combination chemotherapy with etoposide plus cisplatin or carboplatin showed disease control rates (DCRs) of overall 74%, with a short median progression-free survival (PFS) of 7 or 5 months, respectively. DCR and PFS for PRRT were 89% and 22 months when administered as monotherapy, versus 100% and 27 months upon combination with somatostatin analog (SSA) therapy. Of note, PRRT also achieved disease control as best response in 5/5 (100%) selected cases of NEC G3. Conclusion. Further prospective studies are warranted to help stratify available options for therapeutic intervention in NEN patients.

Background Delayed gastric emptying (DGE) remains the most frequent complication following pancreatoduodenectomy (PD) with published incidences as high as 61%. The present study investigates the impact of bowel reconstruction techniques on DGE following classic PD (Whipple-Kausch procedure) with pancreatogastrostomy (PG). Methods We included 168 consecutive patients who underwent PD with PG with either Billroth II type (BII, n  = 78) or Roux-en-Y type reconstruction (ReY, n  = 90) between 2004 and 2015. Excluded were patients with conventional single loop reconstruction after pylorus preserving procedures. DGE was classified according to the 2007 International Study Group of Pancreatic Surgery definition. Patients were analyzed regarding severity of DGE, morbidity and mortality, length of hospital stay and demographic factors. Results No difference was observed between BII and ReY regarding frequency of DGE. Overall rate for clinically relevant DGE was 30% (ReY) and 26% (BII). BII and ReY did not differ in terms of demographics, morbidity or mortality. DGE significantly prolongs ICU (four vs. two days) and hospital stay (20.5 vs. 14.5 days). Risk factors for DGE development are advanced age, retrocolic reconstruction, postoperative hemorrhage and major complications. Conclusions The occurrence of DGE can not be influenced by the type of alimentary reconstruction (ReY vs. BII) following classic PD with PG. Old age and major complications could be identified as important risk factors in multivariate analysis. Trial registration German Clinical Trials Register (DRKS) DRKS00011860 . Registered 14 March 2017.

PurposeHepatic resection is established as a safe procedure for colorectal, neuroendocrine, and sarcoma liver metastases. The present study evaluates whether liver resection is an option for patients with non-colorectal, non-neuroendocrine, and non-sarcoma metastases of the liver.MethodsAccording to data from our prospective clinical tumor registry, we reviewed the medical records of 44 consecutive patients with non-colorectal, non-neuroendocrine, and non-sarcoma liver metastases, who underwent hepatic resection from January 2000 to December 2008. Univariate Kaplan–Meier analysis and a stepwise multivariable Cox regression model were applied.ResultsFollowing hepatic resection, mean overall survival was 21 months, and 5-year survival was 20%. Following hepatic resection, gender, histology, and chemotherapy were of prognostic value in our patient cohort in univariate analysis (p < 0.05). Multivariate survival analysis confirmed chemotherapy (p = 0.002) as an independent prognostic variable. Following initial resection of the primary tumor, synchrone occurrence of metastases, histology, localization of primary, perioperative complications, interval between initial resection of the primary tumor and resection of the metastases, and metastases in follow-up after hepatic resection were of prognostic value in univariate analysis (p < 0.05). Histology (p = 0.017) and interval between resection of the primary and resection of the metastases (p = 0.030) were confirmed as independent prognostic variables in multivariate survival analysis.ConclusionsHepatic resection seems to be a safe and promising additive for a selective group of patients with non-colorectal, non-neuroendocrine, and non-sarcoma metastases of the liver.