Birgitte Viuff

The acute phase response of haptoglobin and serum amyloid A (SAA) in cattle undergoing experimental infection with bovine respiratory syncytial virus.

Abstract The ability of a pure virus infection to induce an acute phase protein response is of interest as viral infections are normally considered to be less efficient in inducing an acute phase protein response than bacterial infections. This was studied in a bovine model for infection with bovine respiratory syncytial virus (BRSV), analysing the induction of the two most dominant bovine acute phase proteins haptoglobin and serum amyloid A (SAA). Strong and reproducible acute phase responses were detected for both proteins, peaking at around 7–8 days after inoculation of BRSV, while no response was seen in mock-inoculated control animals. The serum concentrations reached for SAA and haptoglobin during the BRSV-induced acute phase response were generally the same or higher than previously reported for bacterial infections in calves. The magnitude and the duration of the haptoglobin response was found to correlate well with the severity of clinical signs (fever) and with the extent of lung consolidation while SAA responded most rapidly to infection.

Replication and Clearance of Respiratory Syncytial Virus : Apoptosis Is an Important Pathway of Virus Clearance after Experimental Infection with Bovine Respiratory Syncytial Virus

Human respiratory syncytial virus is an important cause of severe respiratory disease in young children, the elderly, and in immunocompromised adults. Similarly, bovine respiratory syncytial virus (BRSV) is causing severe, sometimes fatal, respiratory disease in calves. Both viruses are pneumovirus and the infections with human respiratory syncytial virus and BRSV have similar clinical, pathological, and epidemiological characteristics. In this study we used experimental BRSV infection in calves as a model of respiratory syncytial virus infection to demonstrate important aspects of viral replication and clearance in a natural target animal. Replication of BRSV was demonstrated in the luminal part of the respiratory epithelial cells and replication in the upper respiratory tract preceded the replication in the lower respiratory tract. Virus excreted to the lumen of the respiratory tract was cleared by neutrophils whereas apoptosis was an important way of clearance of BRSV-infected epithelial cells. Neighboring cells, which probably were epithelial cells, phagocytized the BRSV-infected apoptotic cells. The number of both CD4+ and CD8+ T cells increased during the course of infection, but the T cells were not found between the epithelial cells of the bronchi up until apoptosis was no longer detected, thus in the bronchi there was no indication of direct contact-dependent T-cell-mediated cytotoxicity in the primary infection.

Distribution of sialic acid receptors and influenza A virus of avian and swine origin in experimentally infected pigs

BackgroundPigs are considered susceptible to influenza A virus infections from different host origins because earlier studies have shown that they have receptors for both avian (sialic acid-alpha-2,3-terminal saccharides (SA-alpha-2,3)) and swine/human (SA-alpha-2,6) influenza viruses in the upper respiratory tract. Furthermore, experimental and natural infections in pigs have been reported with influenza A virus from avian and human sources.MethodsThis study investigated the receptor distribution in the entire respiratory tract of pigs using specific lectins Maackia Amurensis (MAA) I, and II, and Sambucus Nigra (SNA). Furthermore, the predilection sites of swine influenza virus (SIV) subtypes H1N1 and H1N2 as well as avian influenza virus (AIV) subtype H4N6 were investigated in the respiratory tract of experimentally infected pigs using immunohistochemical methods.ResultsSIV antigen was widely distributed in bronchi, but was also present in epithelial cells of the nose, trachea, bronchioles, and alveolar type I and II epithelial cells in severely affected animals. AIV was found in the lower respiratory tract, especially in alveolar type II epithelial cells and occasionally in bronchiolar epithelial cells. SA-alpha-2,6 was the predominant receptor in all areas of the respiratory tract with an average of 80-100% lining at the epithelial cells. On the contrary, the SA-alpha-2,3 was not present (0%) at epithelial cells of nose, trachea, and most bronchi, but was found in small amounts in bronchioles, and in alveoli reaching an average of 20-40% at the epithelial cells. Interestingly, the receptor expression of both SA-alpha-2,3 and 2,6 was markedly diminished in influenza infected areas compared to non-infected areas.ConclusionsA difference in predilection sites between SIV and AIV virus was found, and this difference was in accordance with the distribution of the SA-alpha-2,6 and SA-alpha-2,3 receptor, respectively. The results indicated that the distribution of influenza A virus receptors in pigs are similar to that of humans and therefore challenge the theory that the pig acts as a mixing vessel between human and avian influenza viruses. Furthermore, it was shown that AIV prefers to infect alveolar type II epithelial cells in pigs. This corresponds with findings in humans emphasising the resemblance between the two species.

Sites of Replication of Bovine Respiratory Syncytial Virus in Naturally Infected Calves as Determined by In Situ Hybridization

Replication of bovine respiratory syncytial virus (BRSV) was studied in three naturally infected calves by in situ hybridization using strand-specific RNA probes. One of the calves was a 5-month-old Friesian, the other two calves were a 3-month-old and a 3-week-old Jersey. Two Jersey calves, 3 months and 3 weeks of age, served as controls. Replication of BRSV took place in the luminal lining of the respiratory tract. In one of the BRSV infected animals (calf No. 1), replication was especially seen in the bronchi, whereas in the two other animals (calf Nos. 2 and 3) replication of BRSV was demonstrated in the bronchiolar epithelial cells and in alveolar cells. Syncytia were often observed in the bronchiolar walls and in alveoli and such syncytia were always replicating BRSV. By immunohistochemistry it was possible to demonstrate BRSV antigen at the same location as replication of BRSV was detected. In tissue outside the respiratory tract neither BRSV antigen nor replication of BRSV could be demonstrated.

Antibody dynamics in BRSV-infected Danish dairy herds as determined by isotype-specific immunoglobulins.

Abstract Using specific ELISAs, antibody levels of four different isotypes to bovine respiratory syncytial virus (BRSV) were determined in calves, following experimental BRSV infection. Most calves experienced an increase in the specific IgM and IgG1 titres about 6–10 days after infection with BRSV. The IgM titre was transient showing positive titres for only 5–10 days, while specific IgG1 was present for a longer time. IgA was detected concomitantly with IgM but at a lower level. Production of IgG2 anti-BRSV antibodies was detected from 3 weeks after infection. In two closed herds, repeated blood samplings were performed on young stock to analyse maternal immunity. The passively transferred antibodies were mainly of the IgG1 isotype and the half-life of IgG1 to BRSV was estimated to be 26.6 days. One of the herds had an outbreak of enzootic pneumonia, diagnosed to be caused by BRSV. Furthermore, another herd with acute BRSV was followed by weekly blood samples in six calves; in both herds IgM and IgG1 was detected shortly after the appearance of clinical signs. Serum samples from 50 Danish dairy herds (453 samples) were tested for immunoglobulins of the isotypes IgG1, IgG2 and IgM. The presence of antibodies to BRSV was widespread and more than 54% of the samples had BRSV antibodies of both the IgG1 and IgG2 isotypes indicating a high herd prevalence to BRSV. Test samples from two herds out of 50 were free from all isotypes to BRSV.

An experimental infection model for reproduction of calf pneumonia with bovine respiratory syncytial virus (BRSV) based on one combined exposure of calves

Abstract Bovine respiratory syncytial virus (BRSV) has been recognised as an important pathogen in calf pneumonia for 30 years, but surprisingly few effective infection models for studies of the immune response and the pathogenesis in the natural host have been established. We present a reproducible experimental infection model for BRSV in 2–5-month-old, conventionally reared Jersey calves. Thirty-four colostrum-fed calves were inoculated once by aerosol and intratracheal injection with BRSV. Respiratory disease was recorded in 91% of the BRSV-inoculated calves, 72% had an accompanying rise in rectal temperature and 83% exhibited >5% consolidation of the lung tissue. The disease closely resembled natural outbreaks of BRSV-related pneumonia, and detection of BRSV in nasal secretions and lung tissues confirmed the primary role of BRSV. Nine mock-inoculated control calves failed to develop respiratory disease. This model is a valuable tool for the study of the pathogenesis of BRSV and for vaccine efficacy studies.

Increased pulmonary secretion of tumor necrosis factor-α in calves experimentally infected with bovine respiratory syncytial virus.

Abstract Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory disease among calves in the Danish cattle industry. An experimental BRSV infection model was used to study the pathogenesis of the disease in calves. Broncho alveolar lung lavage (BAL) was performed on 28 Jersey calves, of which 23 were experimentally infected with BRSV and five were given a mock inoculum. The presence of the cytokine tumor necrosis factor α (TNF-α) in the BAL fluids was detected and quantified by a capture ELISA. TNF-α was detected in 21 of the infected animals. The amount of TNF-α in the BAL fluid of calves killed post inoculation day (PID) 2 and 4 was at the same very low level as in the uninfected control animals. Large amounts of TNF-α were detected on PID 6, maximum levels of TNF-α were reached on PID 7, and smaller amounts of TNF-α were seen on PID 8. The high levels of TNF-α appeared on the days where severe lung lesions and clinical signs were obvious and the amounts of BRSV-antigen were at their greatest. Although Pasteurellaceae were isolated from some of the BRSV-infected calves, calves treated with antibiotics before and through the whole period of the infection, as well as BRSV-infected calves free of bacteria reached the same level of TNF-α as animals from which bacteria were isolated from the lungs. It is concluded that significant quantities of TNF-α are produced in the lungs of the calves on PID 6–7 of BRSV infection. The involvement of TNF-α in the pathogenesis of, as well as the anti-viral immune response against, BRSV infection is discussed.

Establishment and characterization of models of chemotherapy resistance in colorectal cancer: Towards a predictive signature of chemoresistance

Current standard treatments for metastatic colorectal cancer (CRC) are based on combination regimens with one of the two chemotherapeutic drugs, irinotecan or oxaliplatin. However, drug resistance frequently limits the clinical efficacy of these therapies. In order to gain new insights into mechanisms associated with chemoresistance, and departing from three distinct CRC cell models, we generated a panel of human colorectal cancer cell lines with acquired resistance to either oxaliplatin or irinotecan. We characterized the resistant cell line variants with regards to their drug resistance profile and transcriptome, and matched our results with datasets generated from relevant clinical material to derive putative resistance biomarkers. We found that the chemoresistant cell line variants had distinctive irinotecan‐ or oxaliplatin‐specific resistance profiles, with non‐reciprocal cross‐resistance. Furthermore, we could identify several new, as well as some previously described, drug resistance‐associated genes for each resistant cell line variant. Each chemoresistant cell line variant acquired a unique set of changes that may represent distinct functional subtypes of chemotherapy resistance. In addition, and given the potential implications for selection of subsequent treatment, we also performed an exploratory analysis, in relevant patient cohorts, of the predictive value of each of the specific genes identified in our cellular models.

Genetic and biological characterisation of an avian-like H1N2 swine influenza virus generated by reassortment of circulating avian-like H1N1 and H3N2 subtypes in Denmark

BackgroundThe influenza A virus subtypes H1N1, H1N2 and H3N2 are the most prevalent subtypes in swine. In 2003, a reassorted H1N2 swine influenza virus (SIV) subtype appeared and became prevalent in Denmark. In the present study, the reassortant H1N2 subtype was characterised genetically and the infection dynamics compared to an “avian-like” H1N1 virus by an experimental infection study.MethodsSequence analyses were performed of the H1N2 virus. Two groups of pigs were inoculated with the reassortant H1N2 virus and an “avian-like” H1N1 virus, respectively, followed by inoculation with the opposite subtype four weeks later. Measurements of HI antibodies and acute phase proteins were performed. Nasal virus excretion and virus load in lungs were determined by real-time RT-PCR.ResultsThe phylogenetic analysis revealed that the reassorted H1N2 virus contained a European “avian-like” H1-gene and a European “swine-like” N2-gene, thus being genetically distinct from most H1N2 viruses circulating in Europe, but similar to viruses reported in 2009/2010 in Sweden and Italy. Sequence analyses of the internal genes revealed that the reassortment probably arose between circulating Danish “avian-like” H1N1 and H3N2 SIVs. Infected pigs developed cross-reactive antibodies, and increased levels of acute phase proteins after inoculations. Pigs inoculated with H1N2 exhibited nasal virus excretion for seven days, peaking day 1 after inoculation two days earlier than H1N1 infected pigs and at a six times higher level. The difference, however, was not statistically significant. Pigs euthanized on day 4 after inoculation, had a high virus load in all lung lobes. After the second inoculation, the nasal virus excretion was minimal. There were no clinical sign except elevated body temperature under the experimental conditions.ConclusionsThe “avian-like” H1N2 subtype, which has been established in the Danish pig population at least since 2003, is a reassortant between circulating swine “avian-like” H1N1 and H3N2. The Danish H1N2 has an “avian-like” H1 and differs from most other reported H1N2 viruses in Europe and North America/Asia, which have H1-genes of human or “classical-swine” origin, respectively. The variant seems, however, also to be circulating in countries like Sweden and Italy. The infection dynamics of the reassorted “avian-like” H1N2 is similar to the older “avian-like” H1N1 subtype.

MCT1 and MCT4 Expression and Lactate Flux Activity Increase During White and Brown Adipogenesis and Impact Adipocyte Metabolism

Adipose tissue takes up glucose and releases lactate, thereby contributing significantly to systemic glucose and lactate homeostasis. This implies the necessity of upregulation of net acid and lactate flux capacity during adipocyte differentiation and function. However, the regulation of lactate- and acid/base transporters in adipocytes is poorly understood. Here, we tested the hypothesis that adipocyte thermogenesis, browning and differentiation are associated with an upregulation of plasma membrane lactate and acid/base transport capacity that in turn is important for adipocyte metabolism. The mRNA and protein levels of the lactate-H+ transporter MCT1 and the Na+,HCO3− cotransporter NBCe1 were upregulated in mouse interscapular brown and inguinal white adipose tissue upon cold induction of thermogenesis and browning. MCT1, MCT4, and NBCe1 were furthermore strongly upregulated at the mRNA and protein level upon differentiation of cultured pre-adipocytes. Adipocyte differentiation was accompanied by increased plasma membrane lactate flux capacity, which was reduced by MCT inhibition and by MCT1 knockdown. Finally, in differentiated brown adipocytes, glycolysis (assessed as ECAR), and after noradrenergic stimulation also oxidative metabolism (OCR), was decreased by MCT inhibition. We suggest that upregulation of MCT1- and MCT4-mediated lactate flux capacity and NBCe1-mediated HCO3−/pH homeostasis are important for the physiological function of mature adipocytes.