Filling two needs with one deed: a combinatory mucosal vaccine against influenza A virus and respiratory syncytial virus.

Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFß or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.

Single-cell analysis reveals lasting immunological consequences of influenza infection and respiratory immunization in the pig lung.

The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single cell RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leukocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing hemagglutinin and nucleoprotein with or without IL-1ß. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1ß reduced the number of functionally active Treg cells. Second, influenza infection upregulated IFI6 in BAL cells and decreased their susceptibility to virus replication in vitro. Our data provide a reference map of porcine BAL cells and reveal lasting immunological consequences of influenza infection and respiratory immunization in a highly relevant large animal model for respiratory virus infection.

Mucosal tumor vaccination delivering endogenous tumor antigens protects against pulmonary breast cancer metastases.

BACKGROUND: Generally, early-stage breast cancer has a good prognosis. However, if it spreads systemically, especially with pulmonary involvement, prospects worsen dramatically. Importantly, tumor-infiltrating T cells contribute to tumor control, particularly intratumoral T cells with a tissue-resident memory phenotype are associated with an improved clinical outcome. METHODS: Here, we use an adenoviral vector vaccine encoding endogenous tumor-associated antigens adjuvanted with interleukin-1ß to induce tumor-specific tissue-resident memory T cells (TRM) in the lung for the prevention and treatment of pulmonary metastases in the murine 4T1 breast cancer model. RESULTS: The mucosal delivery of the vaccine was highly efficient in establishing tumor-specific TRM in the lung. Concomitantly, a single mucosal vaccination reduced the growth of pulmonary metastases and improved the survival in a prophylactic treatment. Vaccine-induced TRM contributed to these protective effects. In a therapeutic setting, the vaccination induced a pronounced T cell infiltration into metastases but resulted in only a minor restriction of the disease progression. However, in combination with stereotactic radiotherapy, the vaccine increased the survival time and rate of tumor-bearing mice. CONCLUSION: In summary, our study demonstrates that mucosal vaccination is a promising strategy to harness the power of antitumor TRM and its potential combination with state-of-the-art treatments.

Effect of mucosal adjuvant IL-1ß on heterotypic immunity in a pig influenza model.

T cell responses directed against highly conserved viral proteins contribute to the clearance of the influenza virus and confer broadly cross-reactive and protective immune responses against a range of influenza viruses in mice and ferrets. We examined the protective efficacy of mucosal delivery of adenoviral vectors expressing hemagglutinin (HA) and nucleoprotein (NP) from the H1N1 virus against heterologous H3N2 challenge in pigs. We also evaluated the effect of mucosal co-delivery of IL-1ß, which significantly increased antibody and T cell responses in inbred Babraham pigs. Another group of outbred pigs was first exposed to pH1N1 as an alternative means of inducing heterosubtypic immunity and were subsequently challenged with H3N2. Although both prior infection and adenoviral vector immunization induced strong T-cell responses against the conserved NP protein, none of the treatment groups demonstrated increased protection against the heterologous H3N2 challenge. Ad-HA/NP+Ad-IL-1ß immunization increased lung pathology, although viral load was unchanged. These data indicate that heterotypic immunity may be difficult to achieve in pigs and the immunological mechanisms may differ from those in small animal models. Caution should be applied in extrapolating from a single model to humans.

Outcome of clinical experience of introducing a patient with secondary systemic AL-amyloidosis associated with multiple myeloma.

This research is relevant, as AL-amyloidosis refers to a systemic type of disease characterized by aggregation of an improperly folded light chain of an immunoglobulin, mainly in the heart and kidneys, causing organ failure. This study describes the clinical experience of introducing a patient with cardiac amyloidosis associated with multiple myeloma (MM). A clinical case of a patient diagnozed with amyloidosis was considered. Magnetic resonance imaging signs of cardiac amyloidosis were confirmed due to the presence of concentric biventricular hypertrophy without dilation, atrial septal hypertrophy, a tendency to atrial dilation, thickening of valve flaps and atrial walls. Upon admission to the research institute, the patient had an anasarca. More accurate recognition of AL-amyloidosis by cardiologists allows for prescribing earlier treatment and improving results. Conventional treatment of MM and AL-amyloidosis includes a combination of dexamethasone with bortezomib and endoxan. Haematopoietic stem cell transplantation after taking high doses of melphalan has become another treatment option and has led to remission in some patients. The novelty of the study is that an example of a timely complete diagnosis and treatment of a combination of these two diseases was presented, as a result of which the patient has achieved a complete haematological and partial organ response to the underlying disease.

Impact of weathered multi-walled carbon nanotubes on the epithelial cells of the intestinal tract in the freshwater grazers Lymnaea stagnalis and Rhithrogena semicolorata.

Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis (L. stagnalis) and the mayfly Rhithrogena semicolorata (R. semicolorata). In L. stagnalis, bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata, irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs (p < 0.01).

Protective mucosal immunity against SARS-CoV-2 after heterologous systemic prime-mucosal boost immunization.

Several effective SARS-CoV-2 vaccines are currently in use, but effective boosters are needed to maintain or increase immunity due to waning responses and the emergence of novel variants. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic plasmid DNA or mRNA priming result in systemic and mucosal immunity in mice. In contrast to two intramuscular applications of an mRNA vaccine, intranasal boosts with adenoviral vectors induce high levels of mucosal IgA and lung-resident memory T cells (TRM); mucosal neutralization of virus variants of concern is also enhanced. The mRNA prime provokes a comprehensive T cell response consisting of circulating and lung TRM after the boost, while the plasmid DNA prime induces mostly mucosal T cells. Concomitantly, the intranasal boost strategies lead to complete protection against a SARS-CoV-2 infection in mice. Our data thus suggest that mucosal booster immunizations after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses.

Predictive modeling of complex ABO glycan phenotypes by lectin microarrays.

Serological classification of individuals as A, B, O, or AB is a mainstay of blood banking. ABO blood groups or ABH antigens, in addition to other surface glycans, act as unique red blood cell (RBC) signatures and direct immune responses. ABO subgroups present as weakened, mixed field, or unexpected reactivity with serological reagents, but specific designations remain complex. Lectins detect glycan motifs with some recognizing ABH antigens. We evaluated a 45-probe lectin microarray to rapidly analyze ABO blood groups and associated unique glycan signatures within complex biological samples on RBC surface glycoproteins. RBC membrane glycoproteins were prepared from donor RBCs, n = 20 for each blood group. ABO blood group was distinguishable by lectin array, including variations in ABH antigen expression not observed with serology. Principal component analysis highlighted broad ABO blood group clusters with unexpected high and low antigen expression and variations were confirmed with ABH antibody immunoblotting. Using a subset of lectins provided an accurate method to predict an ABO serological phenotype. Lectin microarray highlighted the importance of ABO localization on glycoproteins and glycolipids and pointed to increased glycocalyx complexity associated with the expression of A and B antigens including high mannose and branched polylactosamine. Thus, lectins identified subtle surface ABO blood group glycoprotein density variations not detected by routine serological methods. Transfusion services observe alterations in ABH expression during malignancy, and ABO incompatible solid organ transplantation is not without risk of rejection. The presented methods may identify subtle but clinically significant ABO blood group differences for transfusion and transplantation.

Optically accessible generic exhaust gas test bench for the investigation of fundamental SCR-relevant processes.

In this work, a generic exhaust gas test bench is introduced on which reproducible experiments can be performed to gain a deeper understanding of processes during exhaust gas aftertreatment of internal combustion engines. We present the design and initial flow characterization as well as tomographic measurement results of gaseous water distributions. The aim of the development was to provide a generic geometry as well as highly reproducible process boundary conditions for numerical simulation of exhaust aftertreatment phenomena. The presented initial measurements are intended to demonstrate the qualification of the test bench for extensive experimental characterization ranging from measurements of the spray injection, film evaporation, and reaction kinetics to the highly complex multiphase flow conditions during selective catalytic reduction (SCR) processes, which are characterized by high mass flows and temperatures, pronounced transients, and a corrosive atmosphere.

Cardiovascular risk is associated with a transmural gradient of myocardial oxygenation during adenosine infusion.

AIMS: In patients with coronary artery disease (CAD), a transmural gradient of myocardial perfusion has been repeatedly observed, with the subendocardial layer showing more pronounced perfusion deficits. Oxygenation-sensitive cardiovascular magnetic resonance (OS-CMR) allows for monitoring transmural changes of myocardial oxygenation in vivo. We hypothesized that OS-CMR could help identify a transmural oxygenation gradient as a disease marker in patients at risk for CAD. METHODS AND RESULTS: We assessed 34 patients with known CAD and 28 subjects with coronary risk factors but no evidence of significant CAD. Results were compared with 11 healthy volunteers. OS-CMR was performed at 1.5 T, applying a T2*-weighted cine steady state free precession sequence at baseline and during infusion of adenosine. A reader blinded to patient data quantified the relative change of myocardial oxygenation in OS-CMR, defined by the change of signal intensity (ΔSI%) between baseline and during adenosine infusion in the entire myocardium, the subepicardial layer, and the subendocardial layer. SI changes were homogenous throughout the myocardium in healthy subjects, whereas both, patients with risk factors only and patients with CAD, had a significantly smaller ΔSI% in the subendocardial layer than in the subendocardial layer. Both patient groups had an overall decreased ΔSI% across all layers when compared with healthy subjects (P < 0.05). CONCLUSION: Even in the absence of overt CAD, cardiovascular risk factors are associated with a transmural gradient of the myocardial oxygenation response to adenosine as assessed by OS-CMR. An inducible transmural oxygenation gradient may serve as a non-invasive marker for cardiovascular risk.

Limited post-chemotherapy retroperitoneal resection of residual tumour in non-seminomatous testicular cancer: complications, outcome and quality of life.

BACKGROUND: Resection of residual masses after chemotherapy plays a crucial role in management of patients with germ cell tumours (GCTs). The extent of surgery is controversial and we present the experiences from Aarhus University Hospital over a 20-year period. The aim was to evaluate survival, complications, working ability and quality of life (QOL) following a limited surgical procedure performed to resect residual masses in non-seminomatous testicular cancer patients after chemotherapy. MATERIAL AND METHODS: A consecutive patient cohort of 109 patients having surgery between 1993 and 2013 was investigated. Hospital records were reviewed and complications were graded according to the Clavien-Dindo classification. QOL data were assessed in a cross-sectional analysis using the European Organisation for Research and Treatment of Cancer (EORTC), QLQ-C30 version 3.0. Patients were matched 1:1 with controls to evaluate the influence of surgical resection on the QOL. RESULTS: With a median follow-up of 10.3 years, 11 relapses in retroperitoneum were recorded in 10 patients (9%), and four patients (5%) died of disease progression. The majority of relapses in patients considered having no evidence of disease (NED) after primary retroperitoneal surgery occurred 10 + years after treatment and was outside the field of the elective lymph node dissection. Twenty-seven (44%) grade I, 15 (24%) grade II, 7 (11%) grade IIIa and 13 (21%) grade IIIb complications were recorded. No grade IV and V complications were observed. Twenty-three patients (20%) reported loss of antegrade ejaculation. We found no significant differences between patients and controls regarding QOL. CONCLUSION: The survival outcome and complication rate are favourable and are comparable to studies involving full and modified template lymph node dissection. We find that limited resection constitutes an applicable and safe procedure. Limited surgical resection did not influence the patients' long-term QOL. Longer follow-up might be considered.

Tracking the origin of simultaneous endometrial and ovarian cancer by next-generation sequencing - a case report.

BACKGROUND: Endometrioid adenocarcinoma of the uterus and ovarian endometrioid carcinoma share many morphological and molecular features. Differentiation between simultaneous primary carcinomas and ovarian metastases of an endometrial cancer may be very challenging but is essential for prognostic and therapeutic considerations. CASE PRESENTATION: In the present case study of a 33 year-old patient we used targeted amplicon next-generation re-sequencing for clarifying the origin of synchronous endometrioid cancer of the corpus uteri and the left ovary. The patient developed a metachronous lung metastasis of an endometrioid adenocarcinoma four years after hyster- and adnexectomy, vaginal brachytherapy and treatment with the synthetic steroid tibolone. Removal of the metastasis and megestrol treatment for seven years led to a complete remission. A total of 409 genes from the Ampliseq Comprehensive Cancer Panel (Ion Torrent, Thermo Fisher) were analysed by next generation sequencing and mutations in 10 genes, including ARID1A, CTNNB1, PIK3CA and PTEN were identified and confirmed by Sanger sequencing. Primary endometrial as well as ovarian cancer showed an identical mutational profile, suggesting the presence of an ovarian metastasis of the endometrial cancer, rather than a simultaneous endometrial and ovarian cancer. The metachronous lung metastasis showed a different mutational profile compared to the primary cancer. Immunohistochemical staining of the corresponding proteins suggested that the tumour development was driven by alterations in the protein function rather than by changes of the protein abundance in the cell. CONCLUSIONS: Our results have demonstrated next generation sequencing as a valuable tool in the differentiation of synchronous primary tumours and metastases, which has an important impact on the clinical decision making process. Similar to breast cancer, targeted therapies based on mutational tumour profiling will become increasingly important in endometrial and ovarian cancer. In summary, our results support the usage of next generation sequencing as a supplementary diagnostic tool, assisting in personalized precision medicine.

After initial treatment for primary breast cancer: information needs, health literacy, and the role of health care workers.

PURPOSE: After a short hospital stay of just some days follows long-term outpatient care for breast cancer patients. The aim of the study is to describe the information needs of breast cancer outpatients and to get in touch with aspects of health literacy, as well as contact various health care workers. METHODS: In a multicenter study, patients were asked about their information needs 10 weeks after surgery. The analysis on hand includes data about 1248 female patients. In addition to descriptive analyses identifying the most prevalent information needs, logistic regression analyses were calculated to identify factors associated with these. RESULTS: The results show that information needs of breast cancer outpatients are mainly in "follow-up after acute treatment", "coping with long-term side effects", and "heredity of breast cancer". In addition to sociodemographic patient characteristics, perceived helpful contacts with various health care workers as well as a satisfactory patient's level of health literacy reduced the probability of unmet information needs. CONCLUSIONS: Breast cancer outpatients have numerous information needs. In addition to provide information at the right time regarding a specific disease phase, it is important that health professionals' support affected breast cancer patients in coping with the new situation.

The Influence of Health Literacy on Information Needs Among Women Newly Diagnosed With Breast Cancer, With Special Reference to Employment Status.

Breast cancer is the most frequent type of malignancy among women throughout Germany. The present analysis aimed to identify information needs and aspects of health literacy in women of working age newly diagnosed with breast cancer. PIAT is a prospective multicenter cohort study in which patients were asked about their information needs at 3 assessment points: postoperatively, after 10 weeks, and after 40 weeks. The present analysis includes data from 1,344 female patients after the first assessment point. In addition to descriptive analyses, logistic regression analyses were calculated. Results of the study show that, in addition to sociodemographic characteristics, the level of health literacy and the employment status of the women who responded to the inquiry influence specific unmet information needs. Most frequently mentioned unmet information needs relate to supplementary naturopathy, nutrition, health-promoting measures, and working during breast cancer. Patients with breast cancer are often provided with large amounts of information during their hospital stay indicating this information is not targeted to patient needs and may be overwhelming. The results show that information on everyday life needs such as supplementary naturopathy were important for the sample newly diagnosed with breast cancer. Employed women in particular have questions regarding working during cancer or tax relief.

The health literate health care organization 10 item questionnaire (HLHO-10): development and validation.

BACKGROUND: While research on individual health literacy is steadily increasing, less attention has been paid to the context of care that may help to increase the patient's ability to navigate health care or to compensate for their limited health literacy. In 2012, Brach et al. introduced the concept of health literate health care organizations (HLHOs) to describe the organizational context of care. This paper presents our effort in developing and validating an HLHO instrument. METHOD: Ten items were developed to represent the ten attributes of HLHO (HLHO-10) based on a literature review, an expert workshop, a focus group discussion, and qualitative interviews. The instrument was applied in a key informant survey in 51 German hospitals as part of a larger study on patient information and training needs (PIAT-study). Item properties were analyzed and a confirmatory factor analysis (CFA) was conducted to test the instrument's unidimensionality. To investigate the instrument's predictive validity, a multilevel analysis was performed that used the HLHO-10 score to predict the adequacy of information provided to 1,224 newly-diagnosed breast cancer patients treated at the sample hospitals. RESULTS: Cronbach's α of the resulting scale was 0.89. CFA verified the one-factor structure after allowing for the correlation for four pairs of error terms. In the multilevel model, HLHO-10 significantly predicted the adequacy of information as perceived by patients. CONCLUSION: The instrument has satisfactory reliability and validity. It provides a useful tool to assess the degree to which health care organizations help patients to navigate, understand, and use information and services. Further validation should include participant observation in health care organizations and a sample that is not limited to breast cancer care.

Acute kidney injury after cardiac surgery: is minocycline protective?

BACKGROUND AND OBJECTIVES: Acute kidney injury (AKI) after cardiac bypass surgery (CABG) is common and carries a significant association with morbidity and mortality. Since minocycline therapy attenuates kidney injury in animal models of AKI, we tested its effects in patients undergoing CABG. DESIGN, SETTING, PARTICIPANTS AND MEASUREMENTS: This is a randomized, double-blinded, placebo-controlled, multi-center study. We screened high risk patients who were scheduled to undergo CABG in two medical centers between Jan 2008 and June 2011. 40 patients were randomized and 19 patients in each group completed the study. Minocycline prophylaxis was given twice daily, at least for four doses prior to CABG. Primary outcome was defined as AKI [0.3 mg/dl increase in creatinine (Cr)] within 5 days after surgery. Daily serum Cr for 5 days, various clinical and hemodynamic measures and length of stay were recorded. RESULTS: The two groups had similar baseline and intra-operative characteristics. The primary outcome occurred in 52.6% of patients in the minocycline group as compared to 36.8% of patients in the placebo group (p = 0.51). Peak Cr was 1.6 ± 0.7 vs. 1.5 ± 0.7 mg/dl (p = 0.45) in minocycline and placebo groups, respectively. Death at 30 days occurred in 0 vs. 10.5% in the minocycline and placebo groups, respectively (p = 0.48). There were no differences in post-operative length of stay, and cardiovascular events between the two groups. There was a trend towards lower diastolic pulmonary artery pressure [16.8 ± 4.7 vs. 20.7 ± 6.6 mmHg (p = 0.059)] and central venous pressure [11.8 ± 4.3 vs. 14.6 ± 5.6 mmHg (p = 0.13)] in the minocycline group compared to placebo on the first day after surgery. CONCLUSIONS: Minocycline did not protect against AKI post-CABG.

Adjuvant granulocyte colony-stimulating factor therapy results in improved spatial learning and stimulates hippocampal neurogenesis in a mouse model of pneumococcal meningitis.

Despite the development of new antibiotic agents, mortality of pneumococcal meningitis remains high. In addition, meningitis results in severe long-term morbidity, most prominently cognitive deficits. Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of hematopoietic progenitor cells and increases the number of circulating neutrophil granulocytes. This study investigated the effect of adjuvant G-CSF treatment on cognitive function after pneumococcal meningitis. C57BL/6 mice were infected by subarachnoid injection of Streptococcus pneumoniae serotype 3 and treated with ceftriaxone and G-CSF subcutaneously or ceftriaxone alone for 5 days. Clinical scores, motor performance, and mortality during bacterial meningitis were unaffected by adjuvant G-CSF treatment. No effect of G-CSF treatment on production of proinflammatory cytokines or activation of microglia or astrocytes was observed. The G-CSF treatment did, however, result in hippocampal neurogenesis and improved spatial learning performance 6 weeks after meningitis. These results suggest that G-CSF might offer a new adjuvant therapeutic approach in bacterial meningitis to reduce long-term cognitive deficits.

E- and p-selectins are essential for repopulation of chronic myelogenous and chronic eosinophilic leukemias in a scid mouse xenograft model.

In chronic myelogenous (CML) and chronic eosinophilic leukemia (CEL), neoplastic cells spread via the circulation into various extramedullary organs. As E- and P-selectin constitute the starting point for the leucocyte adhesion/invasion cascade, and CEL and CML cells share many properties with normal granulocytes, we investigated the role of these selectins in CEL and CML cell expansion and organ invasion in a xenotransplantation model using scid mice. Using two human leukemic cell lines (EOL-1 and K562), we were able to show that E- and P-selectins mediate leukemia cell tethering and adherence in a laminar flow assay. While E-selectin binding depended on sialylated carbohydrate moieties, P-selectin binding was completely (K562) or partially (EOL-1) independent of these carbohydrates indicating the involvement of non-canonical selectin ligands. In a xenograft model in scid mice, both cell lines invaded the bone marrow and other organs, formed chloromas, and ultimately produced an overt leukemia. In contrast, in E- and P-selectin knockout scid mice, the cells failed to show engraftment in 8 out of 10 animals and even if they did engraft, they produced only little organ invasion and chloroma formation. Together, these data suggest that E- and P-selectins play an important role in leukemic dissemination in CML and CEL.

Incoming RNA virus nucleocapsids containing a 5'-triphosphorylated genome activate RIG-I and antiviral signaling.

Host defense to RNA viruses depends on rapid intracellular recognition of viral RNA by two cytoplasmic RNA helicases: RIG-I and MDA5. RNA transfection experiments indicate that RIG-I responds to naked double-stranded RNAs (dsRNAs) with a triphosphorylated 5' (5'ppp) terminus. However, the identity of the RIG-I stimulating viral structures in an authentic infection context remains unresolved. We show that incoming viral nucleocapsids containing a 5'ppp dsRNA "panhandle" structure trigger antiviral signaling that commences with RIG-I, is mediated through the adaptor protein MAVS, and terminates with transcription factor IRF-3. Independent of mammalian cofactors or viral polymerase activity, RIG-I bound to viral nucleocapsids, underwent a conformational switch, and homo-oligomerized. Enzymatic probing and superresolution microscopy suggest that RIG-I interacts with the panhandle structure of the viral nucleocapsids. These results define cytoplasmic entry of nucleocapsids as the proximal RIG-I-sensitive step during infection and establish viral nucleocapsids with a 5'ppp dsRNA panhandle as a RIG-I activator.

Selectin binding is essential for peritoneal carcinomatosis in a xenograft model of human pancreatic adenocarcinoma in pfp--/rag2-- mice.

BACKGROUND AND OBJECTIVE: E- and P-selectins expressed on the luminal surface of mesodermally derived endothelial cells play a crucial role in the formation of haematogenous metastases in a number of malignancies. As peritoneal mesothelial cells are also derived form the mesoderm, it was hypothesised that selectins are also of importance in peritoneal tumour spread. METHODS: Immunohistochemistry was used to identify selectin expression on normal human peritoneum and isolated mesothelial cells. E- and P-selectin interactions with human pancreatic adenocarcinoma cells were investigated in dynamic flow assays and flow cytometry; the latter was also used to determine the main selectin ligands on pancreatic adenocarcinoma cell lines PaCa 5061, BxPC-3 and PaCa 5072, and selectin expression on human mesothelial cells. All cell lines were xenografted into the peritoneum of E- and P-selectin-deficient pfp/rag2 mice and selectin wild-type controls. Peritoneal carcinomatosis was quantified using MRI or a scoring system. RESULTS: E- and P-selectin were constitutively expressed on human mesothelial and endothelial cells in the peritoneum. PaCa 5061 and BxPC-3 cells interacted with E- and P-selectins in dynamic flow assays and flow cytometry, with CA19-9 (Sialyl Lewis a) being the main E-selectin ligand. For xenografted PaCa 5061 and BxPC-3 cells, peritoneal metastasis was significantly reduced in E- and P-selectin double knockout mice compared with wild-type pfp/rag2 animals. In contrast, PaCa 5072 cells were almost devoid of selectin binding sites and no intraperitoneal tumour growth was observed. CONCLUSION: Interactions of tumour cells with peritoneal selectins play an important role in the peritoneal spread of pancreatic adenocarcinoma.