The 5' untranslated region of the anti-apoptotic protein Survivin contains an inhibitory upstream AUG codon.

Survivin (BIRC5) is an anti-apoptotic protein that is important in cancer. Mechanisms responsible for controlling Survivin levels in cells include transcriptional regulation and modulation of protein stability via post-translational modifications; however to date, translational control has been poorly studied. Here, we focused particularly on the primary control elements present in the Survivin 5' untranslated region (5'UTR). Bioinformatic analysis of ribosome occupancy on the Survivin 5'UTR revealed the presence of elongating ribosomes upstream of the canonical initiator AUG, suggesting an alternative upstream initiator AUG (uAUG) might exist. This uAUG was found out-of-frame at position -71 and appeared as a conserved element in mammals. RACE analysis revealed different transcriptional start sites for BIRC5, which indicated that translational control by this uAUG is restricted to longer 5'UTR variants. We studied the activity of the uAUG in different cell types by cloning the Survivin 5'UTR DNA sequence (wild-type and mutated variants) upstream of renilla luciferase (RLuc) into a pcDNA3 plasmid. Changes in RLuc activity were determined by luminescence assays and Western blotting. Results showed that when this uAUG was mutated to AUU or AGG in the cloned Survivin 5'UTR, RLuc activity was significantly increased. Similar results were obtained when uAUG was positioned inframe with the RLuc initiator AUG. Immunodetection of Renilla (35 kDa) by Western blotting revealed the presence of a second band (37 kDa approximately) in cells transfected with the Inframe reporter constructs, indicating that the uAUG was functional in our experimental conditions. In conclusion, our experimental data demonstrate the presence of an alternative and inhibitory initiator uAUG in the Survivin 5' UTR. This inhibitory uAUG may help understanding how Survivin expression is downregulated under physiological or pathological conditions.

Modulation of Host Immunity by the Human Metapneumovirus.

Globally, as a leading agent of acute respiratory tract infections in children <5 years of age and the elderly, the human metapneumovirus (HMPV) has gained considerable attention. As inferred from studies comparing vaccinated and experimentally infected mice, the acquired immune response elicited by this pathogen fails to efficiently clear the virus from the airways, which leads to an exaggerated inflammatory response and lung damage. Furthermore, after disease resolution, there is a poor development of T and B cell immunological memory, which is believed to promote reinfections and viral spread in the community. In this article, we discuss the molecular mechanisms that shape the interactions of HMPV with host tissues that lead to pulmonary pathology and to the development of adaptive immunity that fails to protect against natural infections by this virus.

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells.

Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse- and human cell-based studies have shown that hRSV infection prevents naïve T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface-anchored N protein prevented immunological synapse assembly by naive CD4(+) T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell-bilayer interface, suggesting that N protein interferes with pMHC-TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.

Human metapneumovirus infection activates the TSLP pathway that drives excessive pulmonary inflammation and viral replication in mice.

Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infections in children and the elderly. The mechanism by which this virus triggers an inflammatory response still remains unknown. Here, we evaluated whether the thymic stromal lymphopoietin (TSLP) pathway contributes to lung inflammation upon hMPV infection. We found that hMPV infection promotes TSLP expression both in human airway epithelial cells and in the mouse lung. hMPV infection induced lung infiltration of OX40L(+) CD11b(+) DCs. Mice lacking the TSLP receptor deficient mice (tslpr(-/-) ) showed reduced lung inflammation and hMPV replication. These mice displayed a decreased number of neutrophils as well a reduction in levels of thymus and activation-regulated chemokine/CCL17, IL-5, IL-13, and TNF-α in the airways upon hMPV infection. Furthermore, a higher frequency of CD4(+) and CD8(+) T cells was found in tslpr(-/-) mice compared to WT mice, which could contribute to controlling viral spread. Depletion of neutrophils in WT and tslpr(-/-) mice decreased inflammation and hMPV replication. Remarkably, blockage of TSLP or OX40L with specific Abs reduced lung inflammation and viral replication following hMPV challenge in mice. Altogether, these results suggest that activation of the TSLP pathway is pivotal in the development of pulmonary pathology and pulmonary hMPV replication.

Understanding Lung Immunopathology Caused by the Human Metapneumovirus: Implications for Rational Vaccine Design.

Acute respiratory tract infections (ARTIs) are the major cause of child mortality worldwide. The human metapneumovirus (hMPV) is one of the leading causes of child hospitalizations due to pneumonia. The adaptive immune response generated by the host against hMPV is usually inefficient at protecting from reinfections, which is repeat throughout life, from childhood to old age. Despite considerable research efforts, to date there are no licensed vaccines to prevent respiratory disease caused by hMPV infection. In this article we review current vaccine strategies tested in animal models and the implication of such studies in understanding the different immune cell populations that contribute to hMPV clearance and the prevention and resolution of lung inflammation upon exposure to the virus.

Immunization with a recombinant bacillus Calmette-Guerin strain confers protective Th1 immunity against the human metapneumovirus.

Along with the human respiratory syncytial virus (hRSV), the human metapneumovirus (hMPV) is one of the leading causes of childhood hospitalization and a major health burden worldwide. Unfortunately, owing to an inefficient immunological memory, hMPV infection provides limited immune protection against reinfection. Furthermore, hMPV can induce an inadequate Th2 type immune response that causes severe lung inflammation, leading to airway obstruction. Similar to hRSV, it is likely that an effective clearance of hMPV would require a balanced Th1 type immunity by the host, involving the activation of IFN-γ-secreting T cells. A recognized inducer of Th1 immunity is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which has been used in newborns for many decades and in several countries as a tuberculosis vaccine. We have previously shown that immunization with BCG strains expressing hRSV Ags can induce an efficient immune response that protects against this virus. In this study, we show that immunization with rBCG strains expressing the phosphoprotein from hMPV also can induce protective Th1 immunity. Mice immunized with rBCG were protected against weight loss, airway inflammation, and viral replication in the lungs after hMPV infection. Our rBCG vaccine also induced the activation of hMPV-specific T cells producing IFN-γ and IL-2, which could protect from hMPV infection when transferred to recipient mice. These data strongly support the notion that rBCG induces protective Th1 immunity and could be considered as an efficient vaccine against hMPV.

Elevated IL-3 and IL-12p40 levels in the lower airway of infants with RSV-induced bronchiolitis correlate with recurrent wheezing.

Respiratory Syncytial Virus (RSV) is the first cause of hospitalization due to bronchiolitis in infants. RSV bronchiolitis has been linked to asthma and recurrent wheezing, however the mechanisms behind this association have not been elucidated. Here, we evaluated the cytokine and chemokine profiles in the airways in infants with RSV bronchiolitis. Nasopharyngeal Aspirates (NPA) and Bronchoalveolar Lavage Fluids (BALF) from infants hospitalized due to RSV bronchiolitis and healthy controls were analyzed for cytokine and chemokine production. We observed elevated levels of Th2 cytokines (IL-3, IL-4, IL-10 and IL-13), pro-inflammatory cytokines and chemokines (IL-1ß, IL-6, TNF-ß, MCP-1/CCL2, MIP-1α/CCL3 and IL-8/CXCL8) in BALF from infants with RSV bronchiolitis, as compared to controls. We found a direct correlation of IL-3 and IL-12p40 levels with the development of recurrent wheezing later in life. These results suggest that IL-3 and IL-12p40 could be considered as molecular predictors for recurrent wheezing due to RSV infection.

Recombinant BCG Vaccines Reduce Pneumovirus-Caused Airway Pathology by Inducing Protective Humoral Immunity.

The Human Respiratory Syncytial Virus (hRSV) and the Human Metapneumovirus (hMPV) are two pneumoviruses that are leading agents causing acute lower respiratory tract infections (ALRTIs) affecting young infants, the elderly, and immunocompromised patients worldwide. Since these pathogens were first discovered, many approaches for the licensing of safe and effective vaccines have been explored being unsuccessful to date. We have previously described that immunization with recombinant strains of Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) expressing the hRSV nucleoprotein (rBCG-N) or the hMPV phosphoprotein (rBCG-P) induced immune protection against each respective virus. These vaccines efficiently promoted viral clearance without significant lung damage, mainly through the induction of a T helper 1 cellular immunity. Here we show that upon viral challenge, rBCG-immunized mice developed a protective humoral immunity, characterized by production of antibodies specific for most hRSV and hMPV proteins. Further, isotype switching from IgG1 to IgG2a was observed in mice immunized with rBCG vaccines and correlated with an increased viral clearance, as compared to unimmunized animals. Finally, sera obtained from animals immunized with rBCG vaccines and infected with their respective viruses exhibited virus neutralizing capacity and protected naïve mice from viral replication and pulmonary disease. These results support the notion that the use of rBCG strains could be considered as an effective vaccination approach against other respiratory viruses with similar biology as hRSV and hMPV.

Evaluation of monoclonal antibodies that detect conserved proteins from Respiratory Syncytial Virus, Metapneumovirus and Adenovirus in human samples.

Human Respiratory Syncytial Virus (hRSV), human Metapneumovirus (hMPV) and Adenovirus (ADV), are three of the most prevalent viruses responsible for pneumonia and bronchiolitis in children and elderly worldwide, accounting for a high number of hospitalizations annually. Diagnosis of these viruses is required to take clinical actions that allow an appropriate patient management. Thereby, new strategies to design fast diagnostic methods are highly required. In the present work, six monoclonal antibodies (mAbs, two for each virus) specific for conserved proteins from hRSV, hMPV and ADV were generated and evaluated through different immunological techniques, based on detection of purified protein, viral particles and human samples. In vitro evaluation of these antibodies showed higher specificity and sensitivity than commercial antibodies tested in this study. These antibodies were used to design a sandwich ELISA tests that allowed the detection of hRSV, hMPV, and ADV in human nasopharyngeal swabs. We observed that hRSV and ADV were detected with sensitivity and specificity equivalent to a current Direct Fluorescence Assay (DFA) methodology. However, hMPV was detected with more sensitivity than DFA. Our data suggest that these new mAbs can efficiently identify infected samples and discriminate from patients infected with other respiratory pathogens.

Role of Salmonella enterica exposure in Chilean Crohn's disease patients.

AIM: To study the association between exposure to Salmonella enterica (SE) and Crohn's disease (CD) and its clinical implications in Chilean patients. METHODS: Ninety-four unrelated Chilean CD patients from CAREI (Active Cohort Registry of Inflammatory Bowel Disease) presenting to a single inflammatory bowel disease (IBD) unit of a University Hospital were prospectively included in this study. A complete clinical evaluation, including smoking history, was performed at the initial visit, and all the important data of clinical evolution of CD were obtained. Blood samples from these CD patients and 88 healthy sex- and age-matched control subjects were analyzed for exposure to SE and for their NOD2/CARD15 gene status using the presence of anti-Salmonella lipopolysaccharide antibodies [immunoglobulin-G type (IgG)] and polymerase chain reaction (PCR), respectively. We also evaluated exposure to SE in 90 sex- and age-matched patients without CD, but with known smoking status (30 smokers, 30 non-smokers, and 30 former smokers). RESULTS: CD patients comprised 54 females and 40 males, aged 35.5 ± 15.2 years at diagnosis with a mean follow-up of 9.0 ± 6.8 years. CD was inflammatory in 59 patients (62.7%), stricturing in 24 (25.5%) and penetrating in 15 (15.5%). Thirty cases (31.9%) had lesions in the ileum, 29 (30.8%) had ileocolonic lesions, 32 (34.0%) had colonic lesions and 23 (24.4%) had perianal disease. Sixteen CD patients (17%) were exposed to SE compared to 15 (17%) of 88 healthy control subjects (P = 0.8). Thirty-one CD patients (32.9%) were smokers, and 7 (7.4%) were former smokers at diagnosis. In the group exposed to SE, 10 of 16 patients (62.5%) were active smokers compared to 21 of 78 patients (26.9%) in the unexposed group (P = 0.01). On the other hand, 10 of 31 smoking patients (32%) were exposed to SE compared to 5 of 56 nonsmoking patients (9%), and one of the seven former smokers (14%) (P = 0.01). In the group of 90 patients without CD, but whose smoking status was known, there was no difference in exposure to SE [3 of 30 smokers (10%), 5 of 30 non-smokers (16%), and 5 of 30 former smokers (16%); P = 0.6]. There were no differences in disease severity between CD patients with and those without anti-SE IgG antibodies, estimated as the appearance of stricturing [2 (12.5%) vs 22 (28.2%); P = 0.2] or penetrating lesions [2 (12.5%) vs 13 (16.6%); P = 1.0]; or the need for immunosuppressants [11 (68.7%) vs 55 (70.5%); P = 1.0], anti-tumor necrosis factor therapy [1 (6.2%) vs 7 (8.9%); P = 1.0], hospitalization [13 (81.2%) vs 58 (74.3%); P = 0.7], or surgery [3 (18.7%) vs 12 (15.3%); P = 0.3), respectively]. No other factors were associated with SE, including NOD2/CARD15 gene status. Seventeen CD patients (18%) had at least one mutation of the NOD2/CARD15 gene. CONCLUSION: Our study found no association between exposure to SE and CD. We observed a positive correlation between SE exposure and cigarette smoking in Chilean patients with CD, but not with disease severity.

Chromosomal excision of a new pathogenicity island modulates Salmonella virulence in vivo.

Although the excision of unstable pathogenicity islands is a phenomenon that has been described for several virulent bacteria, whether this process directly affects the capacity of these microorganisms to cause disease in their hosts remains unknown. Salmonella enterica serovar Enteritidis (S. Enteritidis) is an enterobacterium that harbors several unstable pathogenicity islands that can excise from the main bacterial chromosome. Here we have evaluated whether excision of one of these pathogenicity islands, denominated as Region of Difference 21 (ROD21), is required for S. Enteritidis to cause disease in the host. By means of genetic targeting of the integrase encoded by the ROD21 we have generated S. Enteritidis strains unable to excise ROD21. The failure to excise ROD21 significantly reduced the capacity to cause a lethal disease and to colonize the spleen and liver of mice, as compared to wild type S. Enteritidis. On the contrary, S. Enteritidis strains overexpressing an excisionase protein increased the frequency of ROD21 excision and showed an improved capacity to cause lethal disease in mice. Accordingly, strains unable to excise ROD21 showed an altered expression of genes located in this pathogenicity island. Our results suggest that the genetic excision of the pathogenicity island ROD21 in S. Enteritidis modulates the capacity of this bacterium to cause disease in mice due to a change in the expression of virulence genes.