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Background Seaweed polysaccharides have been recommended as anticancer supplements and for boosting human health; however, their benefits in the treatment of triple-negative breast cancers (TNBCs) and improving immune surveillance remain unclear. Olaparib is a first-in-class poly (ADP-ribose) polymerase inhibitor. Oligo-Fucoidan, a low-molecular-weight sulfated polysaccharide purified from brown seaweed (Laminaria japonica), exhibits significant bioactivities that may aid in disease management. Methods Macrophage polarity, clonogenic assays, cancer stemness properties, cancer cell trajectory, glucose metabolism, the TNBC 4T1 cells and a 4T1 syngeneic mouse model were used to inspect the therapeutic effects of olaparib and Oligo-Fucoidan supplementation on TNBC aggressiveness and microenvironment. Results Olaparib treatment increased sub-G1 cell death and G2/M arrest in TNBC cells, and these effects were enhanced when Oligo-Fucoidan was added to treat the TNBC cells. The levels of Rad51 and programmed death-ligand 1 (PD-L1) and the activation of epidermal growth factor receptor (EGFR) and adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) facilitate drug resistance and TNBC metastasis. However, the combination of olaparib and Oligo-Fucoidan synergistically reduced Rad51 and PD-L1 levels, as well as the activity of EGFR and AMPK; consistently, TNBC cytotoxicity and stemness were inhibited. Oligo-Fucoidan plus olaparib better inhibited the formation of TNBC stem cell mammospheroids with decreased subpopulations of CD44high/CD24low and EpCAMhigh cells than monotherapy. Importantly, Oligo-Fucoidan plus olaparib repressed the oncogenic interleukin-6 (IL-6)/p-EGFR/PD-L1 pathway, glucose uptake and lactate production. Oligo-Fucoidan induced immunoactive and antitumoral M1 macrophages and attenuated the side effects of olaparib, such as the promotion on immunosuppressive and protumoral M2 macrophages. Furthermore, olaparib plus Oligo-Fucoidan dramatically suppressed M2 macrophage invasiveness and repolarized M2 to the M0-like (F4/80high) and M1-like (CD80high and CD86high) phenotypes. In addition, olaparib- and Oligo-Fucoidan-pretreated TNBC cells resulted in the polarization of M0 macrophages into CD80(+) M1 but not CD163(+) M2 macrophages. Importantly, olaparib supplemented with oral administration of Oligo-Fucoidan in mice inhibited postsurgical TNBC recurrence and metastasis with increased cytotoxic T cells in the lymphatic system and decreased regulatory T cells and M2 macrophages in tumors. Conclusion Olaparib supplemented with natural compound Oligo-Fucoidan is a novel therapeutic strategy for reprogramming cancer stemness, metabolism and the microenvironment to prevent local postsurgical recurrence and distant metastasis. The combination therapy may advance therapeutic efficacy that prevent metastasis, chemoresistance and mortality in TNBC patients.

The development of influenza candidate vaccine viruses (CVVs) for pre-pandemic vaccine production represents a critical step in pandemic preparedness. The multiple subtypes and clades of avian or swine origin influenza viruses circulating world-wide at any one time necessitates the continuous generation of CVVs to provide an advanced starting point should a novel zoonotic virus cross the species barrier and cause a pandemic. Furthermore, the evolution and diversity of novel influenza viruses that cause zoonotic infections requires ongoing monitoring and surveillance, and, when a lack of antigenic match between circulating viruses and available CVVs is identified, the production of new CVVs. Pandemic guidelines developed by the WHO Global Influenza Program govern the design and preparation of reverse genetics-derived CVVs, which must undergo numerous safety and quality tests prior to human use. Confirmation of reassortant CVV attenuation of virulence in ferrets relative to wild-type virus represents one of these critical steps, yet there is a paucity of information available regarding the relative degree of attenuation achieved by WHO-recommended CVVs developed against novel viruses with pandemic potential. To better understand the degree of CVV attenuation in the ferret model, we examined the relative virulence of six A/Puerto Rico/8/1934-based CVVs encompassing five different influenza A subtypes (H2N3, H5N1, H5N2, H5N8, and H7N9) compared with the respective wild-type virus in ferrets. Despite varied virulence of wild-type viruses in the ferret, all CVVs examined showed reductions in morbidity and viral shedding in upper respiratory tract tissues. Furthermore, unlike the wild-type counterparts, none of the CVVs spread to extrapulmonary tissues during the acute phase of infection. While the magnitude of virus attenuation varied between virus subtypes, collectively we show the reliable and reproducible attenuation of CVVs that have the A/Puerto Rico/9/1934 backbone in a mammalian model.

This study sought to improve an existing live attenuated influenza vaccine (LAIV) by including nucleoprotein (NP) from wild-type virus rather than master donor virus (MDV). H7N9 LAIV reassortants with 6:2 (NP from MDV) and 5:3 (NP from wild-type virus) genome compositions were compared with regard to their growth characteristics, induction of humoral and cellular immune responses in mice, and ability to protect mice against homologous and heterologous challenge viruses. Although, in general, the 6:2 reassortant induced greater cell-mediated immunity in C57BL6 mice than the 5:3 vaccine, mice immunized with the 5:3 LAIV were better protected against heterologous challenge. The 5:3 LAIV-induced CTLs also had better in vivo killing activity against target cells loaded with the NP366 epitope of recent influenza viruses. Modification of the genome of reassortant vaccine viruses by incorporating the NP gene from wild-type viruses represents a simple strategy to improve the immunogenicity and cross-protection of influenza vaccines.

A major lesson learned from the public health response to the 2009 H1N1 pandemic was the need to shorten the vaccine delivery timeline to achieve the best pandemic mitigation results. A gap analysis of previous pre‐pandemic vaccine development activities identified possible changes in the Select Agent exclusion process that would maintain safety and shorten the timeline to develop candidate vaccine viruses (CVVs) for use in pandemic vaccine manufacture. Here, we review the biosafety characteristics of CVVs developed in the past 15 years to support a shortened preparedness timeline for A(H5) and A(H7) subtype highly pathogenic avian influenza (HPAI) CVVs. Extensive biosafety experimental evidence supported recent changes in the implementation of Select Agent regulations that eliminated the mandatory chicken pathotype testing requirements and expedited distribution of CVVs to shorten pre‐pandemic and pandemic vaccine manufacturing by up to 3 weeks.

Hepatic microRNAs (miRs) regulate local thyroid hormone (TH) action and TH-related lipid metabolism. We previously found that myricetin effectively ameliorated hepatic steatosis by targeting PPAR signaling pathway, in which the differentially expressed genes were TH-responsive. The present study was designed to explore the mechanism by which myricetin regulated miR-dependent TH action and lipid metabolism on high-fat diet (HFD)-induced hepatic steatosis. C57BL/6J mice were fed a HFD with or without 100 mg kg-1 myricetin by oral gavage for 16 weeks (n = 8 for each group). The results showed that myricetin improved HFD-induced hepatic steatosis, increased serum TH levels and hepatic type 1 deiodinase (DIO1) activities, and elevated energy expenditure in relation to the HFD mice. Meanwhile, myricetin inhibited miR-205 and miR-146b up-regulation induced by HFD, and also up-regulated their targets, Dio1 and thyroid hormone receptor b (TRb) expression, at both the transcriptional and translational levels, accompanied by the regulation of TH responsive lipid metabolism genes. Overexpression or knockdown of miR-205 failed to affect Dio1 mRNA and protein levels in primary mouse hepatocytes. Myricetin directly decreased miR-146b expression in miR-146b mimic-treated hepatocytes to elevate TRb levels. However, the beneficial effects of myricetin on hepatic TH action and lipid metabolism were abolished by TRb siRNA in free fatty acid (FFA)-treated hepatocytes. Our results indicated that myricetin attenuated hepatic steatosis via the miR-146b/TRb pathway and should be considered for the management of NAFLD conditions.

Influenza A(H1) viruses circulating in swine represent an emerging virus threat, as zoonotic infections occur sporadically following exposure to swine. A fatal infection caused by an H1N1 variant (H1N1v) virus was detected in a patient with reported exposure to swine and who presented with pneumonia, respiratory failure, and cardiac arrest. To understand the genetic and phenotypic characteristics of the virus, genome sequence analysis, antigenic characterization, and ferret pathogenesis and transmissibility experiments were performed. Antigenic analysis of the virus isolated from the fatal case, A/Ohio/09/2015, demonstrated significant antigenic drift away from the classical swine H1N1 variant viruses and H1N1 pandemic 2009 viruses. A substitution in the H1 hemagglutinin (G155E) was identified that likely impacted antigenicity, and reverse genetics was employed to understand the molecular mechanism of antibody escape. Reversion of the substitution to 155G, in a reverse genetics A/Ohio/09/2015 virus, showed that this residue was central to the loss of hemagglutination inhibition by ferret antisera raised against a prototypical H1N1 pandemic 2009 virus (A/California/07/2009), as well as gamma lineage classical swine H1N1 viruses, demonstrating the importance of this residue for antibody recognition of this H1 lineage. When analyzed in the ferret model, A/Ohio/09/2015 and another H1N1v virus, A/Iowa/39/2015, as well as A/California/07/2009, replicated efficiently in the respiratory tract of ferrets. The two H1N1v viruses transmitted efficiently among cohoused ferrets, but respiratory droplet transmission studies showed that A/California/07/2009 transmitted through the air more efficiently. Preexisting immunity to A/California/07/2009 did not fully protect ferrets from challenge with A/Ohio/09/2015. IMPORTANCE Human infections with classical swine influenza A(H1N1) viruses that circulate in pigs continue to occur in the United States following exposure to swine. To understand the genetic and virologic characteristics of a virus (A/Ohio/09/2015) associated with a fatal infection and a virus associated with a nonfatal infection (A/Iowa/39/2015), we performed genome sequence analysis, antigenic testing, and pathogenicity and transmission studies in a ferret model. Reverse genetics was employed to identify a single antigenic site substitution (HA G155E) responsible for antigenic variation of A/Ohio/09/2015 compared to related classical swine influenza A(H1N1) viruses. Ferrets with preexisting immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015, demonstrating decreased protection. These data illustrate the potential for currently circulating swine influenza viruses to infect and cause illness in humans with preexisting immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness.

Although cigarette smoke is known to alter immune responses, whether and how CD4 T cells are affected is not well-described. We aimed to characterize how exposure to cigarette smoke extract impacts CD4 T cell effector generation in vitro under Th1-polarizing conditions. Our results demonstrate that cigarette smoke directly acts on CD4 T cells to impair effector expansion by decreasing division and increasing apoptosis. Furthermore, cigarette smoke enhances Th1-associated cytokine production and increases expression of the transcription factor T-bet, the master regulator of Th1 differentiation. Finally, we show that exposure to cigarette smoke extract during priming impairs the ability of effectors to form memory cells. Our findings thus demonstrate that cigarette smoke simultaneously enhances effector functions but promotes terminal differentiation of CD4 T cell effectors. This study may be relevant to understanding how smoking can both aggravate autoimmune symptoms and reduce vaccine efficacy.

Background The emergence of avian influenza A(H7N9) virus in poultry causing zoonotic human infections was reported on March 31, 2013. Development of A(H7N9) candidate vaccine viruses (CVV) for pandemic preparedness purposes was initiated without delay. Candidate vaccine viruses were derived by reverse genetics using the internal genes of A/Puerto/Rico/8/34 (PR8). The resulting A(H7N9) CVVs needed improvement because they had titers and antigen yields that were suboptimal for vaccine manufacturing in eggs, especially in a pandemic situation. Methods Two CVVs derived by reverse genetics were serially passaged in embryonated eggs to improve the hemagglutinin (HA) antigen yield. The total viral protein and HA antigen yields of six egg-passaged CVVs were determined by the BCA assay and isotope dilution mass spectrometry (IDMS) analysis, respectively. CVVs were antigenically characterized by hemagglutination inhibition (HI) assays with ferret antisera. Results Improvement of total viral protein yield was observed for the six egg-passaged CVVs; HA quantification by IDMS indicated approximately a twofold increase in yield of several egg-passaged viruses as compared to that of the parental CVV. Several different amino acid substitutions were identified in the HA of all viruses after serial passage. However, HI tests indicated that the antigenic properties of two CVVs remained unchanged. Conclusions If influenza A(H7N9) viruses were to acquire sustained human-to-human transmissibility, the improved HA yield of the egg-passaged CVVs generated in this study could expedite vaccine manufacturing for pandemic mitigation.

Low-molecular-weight Fucoidan (Oligo-Fucoidan) is a sulfated polysaccharide that has a variety of biological effects and has also been shown to have beneficial health effects. However, the molecular mechanisms underlying the therapeutic effects of Oligo-Fucoidan in patients with cancer remain unclear. Using human colorectal cancer HCT116 cells with (p53+/+) or without (p53−/−) normal p53 expression, we found that Oligo-Fucoidan treatment reduces the occurrence of spontaneous DNA lesions. Etoposide induces double strand DNA breaks. Subsequent administration of Oligo-Fucoidan to etoposide-treated cells promotes p53 accumulation, p21 expression and significant decreases in ataxia-telangiectasia-mutated (ATM), checkpoint kinase 1 (Chk1) and γ-H2AX phosphorylation in p53+/+ cells compared with p53−/− cells. Similarly, co-administration of Oligo-Fucoidan with etoposide inhibits ATM, Chk1 and γ-H2AX phosphorylation, particularly in the presence of p53. Furthermore, Oligo-Fucoidan supplementation increases cancer cell death and attenuates the adverse effects induced by etoposide that decreases production of the pro-inflammatory cytokine IL-6 and chemokine CCL2/MCP-1. Importantly, Oligo-Fucoidan decreases the tumor-promoting M2 macrophages in microenvironment as well as collaborates with p53 and works in combination with etoposide to prevent HCT116 tumorigenicity. Our results first demonstrate that p53 enables Oligo-Fucoidan to effectively inhibit tumor progression, and Oligo-Fucoidan minimizes the side effects of chemotherapy and alters tumor microenvironment.

H7 subtype influenza viruses have demonstrated an ocular tropism in humans, causing conjunctivitis and not respiratory symptoms in many infected individuals. However, the molecular determinants which confer ocular tropism are still poorly understood. Here, we used a murine model of ocular inoculation to demonstrate that H7 influenza viruses are more likely to cause infection following ocular exposure than are non-H7 subtype viruses. We included investigation regarding the potential role of several properties of influenza viruses with murine infectivity following ocular inoculation, including virus lineage, pathogenicity, and HA cleavage site composition. Furthermore, we examined the potential contribution of internal proteins to murine ocular infectivity. These studies establish a link between H7 subtype viruses and the risk of heightened infectivity in a mammalian species following ocular exposure, and support the development of non-traditional inoculation methods and models to best understand the human risk posed by influenza viruses of all subtypes.

Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of ‘universal' influenza vaccine strategies., A major goal of vaccine design is to protect against a broad range of pathogen strains. Here the authors isolate a new broadly neutralizing antibody against influenza haemagglutinin from human memory B cells, and identify mutations that increase and broaden the neutralization towards H5 HA subtype.

The objective of this case study is to present our experience of a surgical approach for vaginal agenesis using an acellular porcine small intestinal submucosa (SIS) graft. The present report involved 2 patients diagnosed as having vaginal agenesis due to Meyer-von-Rokitansky-Küster-Hauser syndrome. The operation procedure involved the creation of a neovaginal tunnel and then a mold wrapped with the SIS graft was placed in the neovagina. The duration of surgery was less than 45 min with minimal blood loss and no operative and postoperative complications. Epithelialization of the neovagina was achieved within 2 months after surgery. The neovagina created with this procedure was the same as that of a normal adult vagina histologically and physiologically. In conclusion, the creation of a neovagina using a SIS graft resulted in a favorable outcome and this approach may be a potential alternative to the management of vaginal agenesis.

Recently, we reported a unique influenza A virus subtype H17N10 from little yellow-shouldered bats. Its neuraminidase (NA) gene encodes a protein that appears to be highly divergent from all known influenza NAs and was assigned as a new subtype N10. To provide structural and functional insights on the bat H17N10 virus, X-ray structures were determined for N10 NA proteins from influenza A viruses A/little yellow-shouldered bat/Guatemala/164/2009 (GU09-164) in two crystal forms at 1.95 Å and 2.5 Å resolution and A/little yellow-shouldered bat/Guatemala/060/2010 (GU10-060) at 2.0 Å. The overall N10 structures are similar to each other and to other known influenza NA structures, with a single highly conserved calcium binding site in each monomer. However, the region corresponding to the highly conserved active site of influenza A N1-N9 NA subtypes and influenza B NA differs substantially. In particular, most of the amino acid residues required for NA activity are substituted, and the putative active site is much wider because of displacement of the 150-loop and 430-loop. These structural features and the fact that the recombinant N10 protein exhibits no, or extremely low, NA activity suggest that it may have a different function than the NA proteins of other influenza viruses. Accordingly, we propose that the N10 protein be termed an NA-like protein until its function is elucidated.

Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.

Human infections with highly pathogenic H5N1 avian influenza viruses continue to occur in many parts of the world and pose a considerable public health threat. With the use of animal models, the identification of virulence determinants has been instrumental in improving our understanding of how these viruses cause severe disease in humans. Two genetically similar H5N1 viruses (A/Thailand/16/2004 and A/Thailand/SP83/2004) exhibit high or low virulence phenotypes, respectively, in multiple animal models. Reassortant viruses were generated from this virus pair and evaluated in ferrets. Each of the polymerase genes of A/Thailand/16/2004 virus individually conferred increased virulence to A/Thailand/SP83/2004 virus while the neuraminidase of the low virulence virus reduced virulence and replication efficiency of the virulent virus in ferrets unless the homologous HA was present. Our results demonstrate that H5N1 virus virulence determinants are polygenic and that there is an important correlation between polymerase adaptation, efficient replication in the host, and virulence.

Although H5N1 influenza viruses have been responsible for hundreds of human infections, these avian influenza viruses have not fully adapted to the human host. The lack of sustained transmission in humans may be due, in part, to their avian-like receptor preference. Here, we have introduced receptor binding domain mutations within the hemagglutinin (HA) gene of two H5N1 viruses and evaluated changes in receptor binding specificity by glycan microarray analysis. The impact of these mutations on replication efficiency was assessed in vitro and in vivo. Although certain mutations switched the receptor binding preference of the H5 HA, the rescued mutant viruses displayed reduced replication in vitro and delayed peak virus shedding in ferrets. An improvement in transmission efficiency was not observed with any of the mutants compared to the parental viruses, indicating that alternative molecular changes are required for H5N1 viruses to fully adapt to humans and to acquire pandemic capability.

The evolution of classical swine influenza viruses receptor specificity preceding the emergence of the 2009 H1N1 pandemic virus was analyzed in glycan microarrays. Classical swine influenza viruses from the α, β, and γ antigenic clusters isolated between 1945 and 2009 revealed a binding profile very similar to that of 2009 pandemic H1N1 viruses, with selectivity for α2-6-linked sialosides and very limited binding to α2-3 sialosides. Despite considerable genetic divergence, the ‘human-like’ H1N1 viruses circulating in swine retained strong binding preference for α2-6 sialylated glycans. Interspecies transmission of H1N1 influenza viruses from swine to humans or from humans to swine has not driven selection of viruses with distinct novel receptor binding specificities. Classical swine and human seasonal H1N1 influenza viruses have conserved specificity for similar α2-6-sialoside receptors in spite of long term circulation in separate hosts, suggesting that humans and swine impose analogous selection pressures on the evolution of receptor binding function.

To better understand the immune response to highly pathogenic avian influenza virus, we compared expression of cytokines in chickens infected with avian influenza virus (A/Vietnam/1203/04) to that in uninfected chickens. Gene expression analyses revealed that influenza disseminated to multiple organs where immune responses could be identified. Among those cytokines influenced by influenza infection were the T helper type (Th)1-associated cytokines interleukin (IL)12 and interferon γ. In addition, a corresponding downregulation of the intracytoplasmic factor GATA3 was identified, whereas the Th2 cytokines IL4 and IL10 did not appear to be impacted by the infection. The inflammatory cytokine IL6 also appeared to be highly upregulated along with type 1 and type 3 interferon. Together, these data indicate that a strong inflammatory and Th1 response occurs after highly pathogenic avian influenza infection in the chicken that has implications for strategies that target the immune system for improving resistance to avian influenza.

Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

Influenza virus, subtype H3N8, was transmitted from horses to greyhound dogs in 2004 and subsequently spread to pet dog populations. The co-circulation of H3N8 viruses in dogs and horses makes bi-directional virus transmission between these animal species possible. To understand the dynamics of viral transmission, we performed virologic surveillance in dogs and horses between 2005 and 2008 in the United States. The genomes of influenza A H3N8 viruses isolated from 36 dogs and horses were sequenced to determine their origin and evolution. Phylogenetic analyses revealed that H3N8 influenza viruses from horses and dogs were monophyletic and distinct. There was no evidence of canine influenza virus infection in horses with respiratory disease or new introductions of equine influenza viruses into dogs in the United States. Analysis of a limited number of equine influenza viruses suggested substantial separation in the transmission of viruses causing clinically apparent influenza in dogs and horses.

We generated influenza virus-like particles (VLPs) containing the wild type (WT) H5 hemagglutinin (HA) from A/Viet Nam/1203/04 virus or a mutant H5 HA with a deletion of the multibasic cleavage motif. VLPs containing mutant H5 HA were found to be as immunogenic as VLPs containing WT HA. A single intramuscular vaccination with either type of H5 VLPs provided complete protection against lethal challenge. In contrast, the recombinant H5 HA vaccine was less immunogenic and vaccination even with a 5 fold higher dose did not induce protective immunity. VLP vaccines were superior to the recombinant HA in inducing T helper type 1 immune responses, hemagglutination inhibition titers, and antibody secreting cells, which significantly contribute to inducing protective immunity after a single dose vaccination. This study provides insights into the potential mechanisms of improved immunogenicity by H5 VLP vaccines as an approach to improve the protective efficacy against potential pandemic viruses.

A recombinant live attenuated influenza virus DeltaH5N1 vaccine with a modified hemagglutinin (HA) and intact neuraminidase genes from A/Vietnam/1203/04 (H5N1) and six remaining genome segments from A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) virus was previously shown to be attenuated in chickens, mice and ferrets. Evaluation of the recombinant H5N1 viruses in mice indicated that three independent factors contributed to the attenuation of the DeltaH5N1 vaccine: the attenuating mutations specified by the AA ca loci had the greatest influence, followed by the deletion of the H5 HA multi-basic cleavage site (MBS), and the constellation effects of the AA genes acting in concert with the H5N1 glycoproteins. Restoring the MBS in the H5 HA of the vaccine virus improved its immunogenicity and efficacy, likely as a consequence of increased virus replication, indicating that removal of the MBS had a deleterious effect on the immunogenicity and efficacy of the DeltaH5N1 vaccine in mice.

This study investigated whether transmissible H5 subtype human-avian reassortant viruses could be generated in vivo. To this end, ferrets were coinfected with recent avian H5N1 (A/Thailand/16/04) and human H3N2 (A/Wyoming/3/03) viruses. Genotype analyses of plaque-purified viruses from nasal secretions of coinfected ferrets revealed that approximately 9% of recovered viruses contained genes from both progenitor viruses. H5 and H3 subtype viruses, including reassortants, were found in airways extending toward and in the upper respiratory tract of ferrets. However, only parental H5N1 genotype viruses were found in lung tissue. Approximately 34% of the recovered reassortant viruses possessed the H5 hemagglutinin (HA) gene, with five unique H5 subtypes recovered. These H5 reassortants were selected for further studies to examine their growth and transmissibility characteristics. Five H5 viruses with representative reassortant genotypes showed reduced titers in nasal secretions of infected ferrets compared to the parental H5N1 virus. No transmission by direct contact between infected and naïve ferrets was observed. These studies indicate that reassortment between H5N1 avian influenza and H3N2 human viruses occurred readily in vivo and furthermore that reassortment between these two viral subtypes is likely to occur in ferret upper airways. Given the relatively high incidence of reassortant viruses from tissues of the ferret upper airway, it is reasonable to conclude that continued exposure of humans and animals to H5N1 alongside seasonal influenza viruses increases the risk of generating H5 subtype reassortant viruses that may be shed from upper airway secretions.

Adaptation of avian influenza viruses for replication and transmission in the human host is believed to require mutations in the hemagglutinin glycoprotein (HA) which enable binding to human α2-6 sialosides and concomitant reduction in affinity for avian α2-3 linked sialosides. Here, we show by glycan microarray analyses that the two mutations responsible for such specificity changes in 1957 H2N2 and 1968 H3N2 pandemic viruses, when inserted into recombinant HAs or intact viruses of some recent avian H5N1 isolates (clade 2.2), impart such attributes. This propensity to adapt to human receptors is primarily dependent on arginine at position 193 within the receptor binding site, as well as loss of a vicinal glycosylation site. already have Widespread occurrence of these susceptible H5N1 clade 2.2 influenza strains has already occurred in Europe, the Middle East, and Africa. Thus, these avian strains should be considered ‘high-risk’, because of their significantly lower threshold for acquiring human receptor specificity and, therefore, warrant increased surveillance and further study.

Genetic and antigenic characterization established the uniqueness of this virus circulating in dogs., In 2004, canine influenza virus subtype H3N8 emerged in greyhounds in the United States. Subsequent serologic evidence indicated virus circulation in dog breeds other than greyhounds, but the virus had not been isolated from affected animals. In 2005, we conducted virologic investigation of 7 nongreyhound dogs that died from respiratory disease in Florida and isolated influenza subtype H3N8 virus. Antigenic and genetic analysis of A/canine/Jacksonville/2005 (H3N8) and A/canine/Miami/2005 (H3N8) found similarity to earlier isolates from greyhounds, which indicates that canine influenza viruses are not restricted to greyhounds. The hemagglutinin contained 5 conserved amino acid differences that distinguish canine from equine lineages. The antigenic homogeneity of the canine viruses suggests that measurable antigenic drift has not yet occurred. Continued surveillance and antigenic analyses should monitor possible emergence of antigenic variants of canine influenza virus.

Avian H7 influenza viruses from both the Eurasian and North American lineage have caused outbreaks in poultry since 2002, with confirmed human infection occurring during outbreaks in The Netherlands, British Columbia, and the United Kingdom. The majority of H7 infections have resulted in self-limiting conjunctivitis, whereas probable human-to-human transmission has been rare. Here, we used glycan microarray technology to determine the receptor-binding preference of Eurasian and North American lineage H7 influenza viruses and their transmissibility in the ferret model. We found that highly pathogenic H7N7 viruses from The Netherlands in 2003 maintained the classic avian-binding preference for α2–3-linked sialic acids (SA) and are not readily transmissible in ferrets, as observed previously for highly pathogenic H5N1 viruses. However, H7N3 viruses isolated from Canada in 2004 and H7N2 viruses from the northeastern United States isolated in 2002–2003 possessed an HA with increased affinity toward α2–6-linked SA, the linkage type found prominently on human tracheal epithelial cells. We identified a low pathogenic H7N2 virus isolated from a man in New York in 2003, A/NY/107/03, which replicated efficiently in the upper respiratory tract of ferrets and was capable of transmission in this species by direct contact. These results indicate that H7 influenza viruses from the North American lineage have acquired sialic acid-binding properties that more closely resemble those of human influenza viruses and have the potential to spread to naïve animals.

Effective annual influenza vaccination requires frequent changes in vaccine composition due to both antigenic shift for different subtype hemagglutinins (HAs) and antigenic drift in a particular HA. Here we present a broadly neutralizing human monoclonal antibody with an unusual binding modality. The antibody, designated CT149, was isolated from convalescent patients infected with pandemic H1N1 in 2009. CT149 is found to neutralize all tested group 2 and some group 1 influenza A viruses by inhibiting low pH-induced, HA-mediated membrane fusion. It promotes killing of infected cells by Fc-mediated antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. X-ray crystallographic data reveal that CT149 binds primarily to the fusion domain in HA2, and the light chain is also largely involved in binding. The epitope recognized by this antibody comprises amino-acid residues from two adjacent protomers of HA. This binding characteristic of CT149 will provide more information to support the design of more potent influenza vaccines., Monoclonal antibodies with broadly neutralizing activity are being developed as potential treatment of influenza infections. Here, the authors describe a broadly neutralizing antibody with an unusual mode of binding to viral haemagglutinin, which has been isolated from patients convalescent from pandemic H1N1 influenza infection.

One of the important lessons learned from the 2009 H1N1 pandemic is that a high yield influenza vaccine virus is essential for efficient and timely production of pandemic vaccines in eggs. The current seasonal and pre-pandemic vaccine viruses are generated either by classical reassortment or reverse genetics. Both approaches utilize a high growth virus, generally A/Puerto Rico/8/1934 (PR8), as the donor of all or most of the internal genes, and the wild type virus recommended for inclusion in the vaccine to contribute the hemagglutinin (HA) and neuraminidase (NA) genes encoding the surface glycoproteins. As a result of extensive adaptation through sequential egg passaging, PR8 viruses with different gene sequences and high growth properties have been selected at different laboratories in past decades. The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously. Here, we use reverse genetics to analyze systematically the growth and HA antigen yield of reassortant viruses with 3 different PR8 backbones. A panel of 9 different HA/NA gene pairs in combination with each of the 3 different lineages of PR8 internal genes (27 reassortant viruses) was generated to evaluate their performance. Virus and HA yield assays showed that the PR8 internal genes influence HA yields in most subtypes. Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production. These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.

Type III protein secretion systems, which are organelles with the capacity to deliver bacterial proteins into host cells, have been adapted to deliver heterologous antigens for vaccine development. A limitation of these antigen delivery systems is that some proteins are not amenable to secretion through this pathway. We show here that proteins from the simian and human immunodeficiency viruses that are not permissive for secretion through aSalmonella entericaserovar Typhimurium type III secretion system can be modified to travel this secretion pathway by introduction of discrete mutations. Proteins optimized for secretion were presented more efficiently via the major histocompatibility complex class I pathway and were able to induce a better immune response.

Background CKS257 ( Salmonella typhimurium SL1344 ΔphoP / phoQΔ aroA Δasd ΔstrA / strB pSB2131) is a live oral vaccine vector expressing HIV Gag. Methods HIV Gag was expressed as a fusion protein of a Salmonella Type III secretion system protein SopE, from a balanced lethal asd -based plasmid. Eighteen healthy adults were given single escalating oral doses of 5 × 10 6 to 1 × 10 10 CFU of CKS257 and were monitored for clinical events, shedding and immune responses. Results Adverse events were mild except at the highest dose. Volunteers shed the organism an average of 5.1 days (range 0–13 days). Eighty-three percent (15/18) of subjects had a mucosal immune response to Salmonella LPS and flagella by IgA ELISPOT assay. Seventy-two percent (13/18) of subjects seroconverted to Salmonella antigens. No volunteer had a response to recombinant Gag as measured by serology, IgA ELISPOT, or immediate ex vivo γ-interferon ELISPOT response to Gag peptide pools. Two volunteers responded to Gag peptides by IL-2 ELISPOT, and 4 of 10 volunteers receiving ≥5 × 10 8 CFU had a response to HIV peptides in a cultured γ-interferon ELISPOT assay. Conclusions Although immunogenicity of the HIV antigen needs augmentation, the attenuated Salmonella strain proved to be an excellent platform for vaccine development.

Bacterial vectors may offer many advantages over other antigen delivery systems for cancer vaccines. We engineered a Salmonella typhimurium vaccine strain to deliver the NY-ESO-1 tumor antigen (S. typhimurium-NY-ESO-1) through a type III protein secretion system. The S. typhimurium-NY-ESO-1 construct elicited NY-ESO-1-specific CD8+ and CD4+ T cells from peripheral blood lymphocytes of cancer patients in vitro. Oral administration of S. typhimurium-NY-ESO-1 to mice resulted in the regression of established NY-ESO-1-expressing tumors. Intratu-moral inoculation of S. typhimurium-NY-ESO-1 to NY-ESO-1-negative tumors resulted in delivery of antigen in vivo and led to tumor regression in the presence of preexisting NY-ESO-1-spedific CD8+ T cells. Specific T cell responses against at least 2 unrelated tumor antigens not contained in the vaccine were observed, demonstrating epitope spreading. We propose that antigen delivery through the S. typhimurium type III secretion system is a promising novel strategy for cancer vaccine development.

Serine proteases have been documented to play key roles in uterine matrix turnover and trophoblastic invasion during implantation. Roles of prostasin serine protease in these processes, however, are currently unclear. The present study was first conducted to investigate the colocalization of prostasin and its cognate serpin, protease nexin-1 (PN-1), in rhesus monkey endometrium and placenta on days 12, 18, and 26 of pregnancy by using in situ hybridization (ISH) and immunohistochemistry. With ISH, expression of prostasin mRNA was intensely localized in the glandular epithelium on days 12 and 18 and in the placental villi, trophoblastic column, trophoblastic shell, and fetal-maternal border on days 18 and 26. With the progress of pregnancy, expression level in the glandular epithelium was significantly decreased, and the accumulation in the placental compartments was further increased. In addition, the stroma and arterioles exhibited modest levels of prostasin signals. However, expression level of PN-1 in these compartments on adjacent sections in the three stages of early pregnancy was weak or below the level of detection. Prostasin protein expression in the endometrium was found to be consistent with the distribution patterns revealed in the ISH experiments. It may be suggested from these results that prostasin is involved in endometrial epithelial morphology establishment, tissue remodeling, and trophoblastic invasion during early pregnancy. The cognate serpin PN-1 was not coordinately expressed along with prostasin, creating a tissue environment favorable for proteolytic activities of prostasin during early pregnancy events.

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.

A novel mutant of bovine viral diarrhea virus (BVDV) was found with a virion assembly phenotype attributable to an insertion into the NS5B polymerase locus. This mutant, termed 5B-741, was engineered by reverse genetics to express NS5B with a C-terminal peptide tag of 22 amino acids. Electroporation of bovine cells with genomic RNA from this mutant showed levels RNA synthesis which were regarded as sufficient for infectivity, yet infectious virions were not produced. Pseudorevertants of mutant 5B-741 that released infectious virions and formed plaques revealed a single nucleotide change (T12369C). This change resulted in a leucine-to-proline substitution within the NS5B tag (L726P). Genetic analysis revealed that indeed a single nucleotide change encoding proline at NS5B position 726 in the pseudorevertant polyprotein mediated recovery of virion assembly function without improving genomic RNA accumulation levels. A subgenomic BVDV reporter replicon (rNS3-5B) was used to analyze the consequences of alterations of the genomic region encoding the NS5B C terminus on replication and assembly. Interestingly, rNS3-5B-L726P (revertant) replicated with the same efficiency as the rNS3-5B-741 mutant but produced 10 times more virions in a trans -packaging assay. These results indicated that impairment of assembly function in 5B-741 was independent of RNA accumulation levels and agreed with the observations from the full-length mutant and revertant genomes. Finally, we recapitulated the packaging defect of 5B-741 with a vaccinia virus expression system to eliminate possible unwanted interactions between the helper virus and the packaged replicon. Taken together, these studies revealed an unexpected role of NS5B in infectious virion assembly.

BACKGROUND The invasion suppressor prostasin is down-regulated in prostate cancer, but the mechanism is unknown. A prostasin-binding protein (PBP) was found in the seminal vesicles, but its identity remains unclear. METHODS Genomic Southern blot analysis using methylation sensitive restriction endonucleases was employed to examine the prostasin gene promoter region in prostate cancer cell lines. RT-PCR was employed to examine prostasin expression under demethylation, histone deacetylase inhibition, and nerve growth factor (NGF) treatment. Liquid column chromatography was employed to purify the PBP from mouse seminal vesicles. The PBP was further characterized by amino acid sequence analysis, recombinant protein expression, protease inhibition and binding assays. Immunohistochemistry and Western blot analysis were used to evaluate PBP expression in the prostate and prostate cancer cells. RESULTS Promoter DNA methylation partly causes the prostasin down-regulation in DU-145 and PC-3 cells, while prostasin expression can be induced by NGF. The PBP is identified to be protease nexin-1 (PN-1), a serpin. PN-1 inhibits prostasin's serine protease activity, is expressed by prostate epithelial cells (PrECs) and prostate cancer cells, and capable of binding to membrane-anchored prostasin. CONCLUSIONS Prostasin's expression and function are regulated by factors in the prostate tissue environment. © 2003 Wiley-Liss, Inc.

The views expressed in this Perspective do not necessarily reflect the views of the journal or of ASM. Zoonotic influenza viruses circulating in poultry and swine pose an ever present threat to human health. In particular, the rapid geographical expansion of highly pathogenic avian influenza (HPAI) A(H5N1) throughout Asia and then into Europe, the Middle East, and Africa during the 2000s galvanized the global community in an attempt to control this rapidly growing threat. Despite successful control efforts in some countries, the virus remains endemic in poultry in at least six countries and continues to cause human illness and deaths as well as countless outbreaks in birds. During the past decade, 668 cases and 393 deaths were detected and reported to the World Health Organization (WHO) (1). During the 17 years since human infections with HPAI A(H5N1) were first identified in Hong Kong, Special Administrative Region, People’s Republic of China, in 1997, these viruses have evolved substantially through mutation and reassortment, resulting in multiple divergent genotypes and clades (2). Ongoing H5N1 circulation has appropriately resulted in a focus on sequencing viral genomes to understand the evolution of these viruses and the significance of observed genetic changes. Expanded laboratory capacity for high-throughput Sanger sequencing and recent technological advances, such as next-generation sequencing and parallel computing, have revolutionized the quantity, quality, and availability of gene sequences and our ability to quickly and accurately analyze these data (3). Consequently, the number of animal and human influenza virus sequences available in publically accessible databases has dramatically increased over the years, as have the bioinformatics …

Dendritic cells (DCs) developmentally regulate antigen uptake by controlling their endocytic capacity. Immature DCs actively internalize antigen. However, mature DCs are poorly endocytic, functioning instead to present antigens to T cells. We have found that endocytic downregulation reflects a decrease in endocytic activity controlled by Rho family GTPases, especially Cdc42. Blocking Cdc42 function by Toxin B treatment or injection of dominant-negative inhibitors of Cdc42 abrogates endocytosis in immature DCs. In mature DCs, injection of constitutively active Cdc42 or microbial delivery of a Cdc42 nucleotide exchange factor reactivates endocytosis. DCs regulate endogenous levels of Cdc42-GTP with activated Cdc42 detectable only in immature cells. We conclude that DCs developmentally regulate endocytosis at least in part by controlling levels of activated Cdc42.

Schwann cells (SCs) differentiate into a myelinating cell when simultaneously adhering to an axon destined for myelination and basal lamina. We are interested in defining the signaling pathway activated by basal lamina. Using SC/sensory neuron (N) cocultures, we identified β1 integrin and F-actin as components of a pathway leading to myelin gene expression and myelination (Fernandez-Valle et al., 1994, 1997). Here, we show that focal adhesion kinase (FAK) and paxillin are constitutively expressed by SCs contacting axons in the absence of basal lamina. Tyrosine phosphorylation of FAK and paxillin increases as SCs form basal lamina and differentiate. FAK and paxillin specifically coimmunoprecipitate with β1 integrin in differentiating SC/N cocultures but not SC-only cultures. Paxillin coimmunoprecipitates with FAK and fyn kinase in differentiating SC/N cocultures. A subset of tyrosine-phosphorylated β1 integrin, FAK, and paxillin molecules reside in the insoluble, F-actin-rich fraction of differentiating cocultures. Cytochalasin D, an actin depolymerizing agent, decreases tyrosine phosphorylation of FAK and paxillin and their association with β1 integrin and causes a dose-dependent increase in the abundance of insoluble FAK and paxillin complexes. Collectively, our work indicates that β1 integrin, FAK, paxillin, and fyn kinase form an actin-associated complex in SCs adhering to basal lamina in the presence of axons. This complex may be important for initiating the process of SC differentiation into a myelinating cell.

S. typhimurium stimulates signaling pathways leading to membrane ruffling, actin cytoskeleton rearrangements, and nuclear responses. The stimulation requires a protein secretion system (type III) that translocates bacterial proteins into the host cell. We show that SopE, a substrate of this secretion system, stimulates cytoskeletal reorganization and JNK activation in a CDC42- and Rac-1-dependent manner. A λgt11 cDNA library screen for proteins that interact with SopE identified Rac-1 and CDC42. Furthermore, purified SopE was shown to stimulate GDP/GTP nucleotide exchange in several Rho GTPases in vitro, including Rac-1 and CDC42. These findings establish a paradigm for microbial stimulation of cellular responses in which the pathogen induces signaling events by directly engaging the signaling machinery within the host cell.

Bat influenza virus H17N10 represents a distinct lineage of influenza A viruses with gene segments coding for proteins that are homologs of the surface antigens, hemagglutinin (HA) and neuraminidase (NA). Our recent study of the N10 NA homolog revealed an NA-like structure, but with a highly divergent putative active site exhibiting little or no NA activity, and provided strong motivation for performing equivalent structural and functional analyses of the H17 HA protein. The overall structure of the H17 HA homolog from A/little yellow-shouldered bat/Guatemala/060/2010 at 3.18 Å resolution is very similar to other influenza HAs, with a putative receptor-binding site containing some conserved aromatic residues that form the base of the sialic acid binding site. However, the rest of the H17 receptor-binding site differs substantially from the other HA subtypes, including substitution of other conserved residues associated with receptor binding. Significantly, electrostatic potential analyses reveal that this putative receptor-binding site is highly acidic, making it unfavorable to bind any negatively charged sialylated receptors, consistent with the recombinant H17 protein exhibiting no detectable binding to sialylated glycans. Furthermore, the fusion mechanism is also distinct; trypsin digestion with recombinant H17 protein, when exposed to pH 4.0, did not degrade the HA1 and HA2, in contrast to other HAs. These distinct structural features and functional differences suggest that the H17 HA behaves very differently compared with other influenza HAs.

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses., Author Summary Previous studies indicated that a novel influenza A virus (H17N10) was circulating in fruit bats from Guatemala (Central America). Herein, we investigated whether similar viruses are present in bat species from South America. Analysis of rectal swabs from bats sampled in the Amazon rainforest region of Peru identified another new influenza A virus from bats that is phylogenetically distinct from the one identified in Guatemala. The genes that encode the surface proteins of the new virus from the flat-faced fruit bat were designated as new subtype H18N11. Serologic testing of blood samples from several species of Peruvian bats indicated a high prevalence of antibodies to the surface proteins. Phylogenetic analyses demonstrate that bat populations from Central and South America maintain as much influenza virus genetic diversity in some gene segments as all other mammalian and avian species combined. The crystal structures of the hemagglutinin and neuraminidase proteins indicate that sialic acid is not a receptor for virus attachment nor a substrate for release, suggesting a novel mechanism of influenza A virus attachment and activation of membrane fusion for entry into host cells. In summary, our findings indicate that bats constitute a potentially important reservoir for influenza viruses.

Cell sheet-mediated tissue regeneration is a promising approach for corneal reconstruction. However, the fragility of bioengineered corneal endothelial cell (CEC) monolayers allows us to take advantage of cross-linked porous gelatin hydrogels as cell sheet carriers for intraocular delivery. The aim of this study was to further investigate the effects of biopolymer concentrations (5–15 wt%) on the characteristic and safety of hydrogel discs fabricated by a simple stirring process combined with freeze-drying method. Results of scanning electron microscopy, porosity measurements, and ninhydrin assays showed that, with increasing solid content, the pore size, porosity, and cross-linking index of carbodiimide treated samples significantly decreased from 508±30 to 292±42 µm, 59.8±1.1 to 33.2±1.9%, and 56.2±1.6 to 34.3±1.8%, respectively. The variation in biopolymer concentrations and degrees of cross-linking greatly affects the Young’s modulus and swelling ratio of the gelatin carriers. Differential scanning calorimetry measurements and glucose permeation studies indicated that for the samples with a highest solid content, the highest pore wall thickness and the lowest fraction of mobile water may inhibit solute transport. When the biopolymer concentration is in the range of 5–10 wt%, the hydrogels have high freezable water content (0.89–0.93) and concentration of permeated glucose (591.3–615.5 µg/ml). These features are beneficial to the in vitro cultivation of CECs without limiting proliferation and changing expression of ion channel and pump genes such as ATP1A1, VDAC2, and AQP1. In vivo studies by analyzing the rabbit CEC morphology and count also demonstrate that the implanted gelatin discs with the highest solid content may cause unfavorable tissue-material interactions. It is concluded that the characteristics of cross-linked porous gelatin hydrogel carriers and their triggered biological responses are in relation to biopolymer concentration effects.

The kallikrein-kinin system participates in blood pressure regulation. One of the kallikrein-kinin system components, kallikrein-binding protein, binds to tissue kallikrein and inhibits its activity in vitro. To investigate potential roles of rat kallikrein-binding protein (RKBP) in vivo, we have developed transgenic mice that express an RKBP gene under the control of the mouse metallothionein metal-responsive promoter. Expression of the transgene, RKBP, was detected in the liver, kidney, lung, heart, pancreas, salivary glands, spleen, brain, testis, and adrenal gland at the mRNA and protein levels. Systolic blood pressures of homozygous transgenic mice were 88.5 +/- 0.8 mm Hg (mean +/- S.E., n = 19, P < 0.001) for one line and 88.8 +/- 1.6 mm Hg (mean +/- S.E., n = 19, P < 0.001) for another, as compared with 100.5 +/- 0.8 mm Hg (mean +/- S.E., n = 18) for control mice. Direct blood pressure measurements of these transgenic mice through an arterial cannula showed similar reductions of blood pressure. Intravenous injection of purified RKBP into mice via a catheter produced a dose-dependent reduction of the mean arterial blood pressure. Our findings suggest that RKBP may function as a vasodilator in vivo, independent of regulating the activity of tissue kallikrein.

We have shown by a variety of microscopical and biochemical techniques that Salmonella spp. are cytotoxic for cultured J774A.1 and bone marrow-derived murine macrophages. The cytotoxicity is initially manifested by inhibition of membrane ruffling and macropinocytosis in infected macrophages, and is followed by cell death. Macrophages killed by Salmonella spp. exhibited features of apoptosis such as condensation and fragmentation of chromatin, membrane blebbing, and the presence of cytoplasmic nucleosomes and apoptotic bodies. Cytotoxicity does not require bacterial internalization as cytochalasin D, a drug that prevents bacterial uptake, did not prevent Salmonella-induced macrophage cell death. However, the cytotoxic effects are strictly dependent upon the expression of the invasion-associated Type III protein-secretion system encoded at centisome 63 of the Salmonella chromosome. Wild-type Salmonella typhimurium grown under conditions that do not allow optical expression of this system or strains of Salmonella carrying mutations in genes that encode components of this protein-secretion system were devoid of macrophage cytotoxicity. In addition, mutations in invJ, spaO, sipB, sipC and sipD, which encode proteins that are secreted via this secretion apparatus and are required for bacterial entry into non-phagocytic cells, also abolished the toxicity. In contrast, mutations in sipA and sptP, which encode secreted proteins that are not required for bacterial invasion, had no effect on macrophage cytotoxicity. These results indicate a close correlation between the mechanisms of bacterial internalization into non-phagocytic cells and those that lead to macrophage cytotoxicity. Host-adapted serotypes of Salmonella such as S. typhi, S. gallinarum and S. dublin were also toxic for murine macrophages, indicating that this virulence property is probably present in most Salmonella spp. and is not associated with the mechanisms responsible for host range.

There is an inverse correlation between systemic blood pressure and urinary kallikrein levels in humans and hypertensive animal models, suggesting that the tissue kallikrein-kinin system plays an important role in blood pressure regulation. In this study, we explored the potential of human kallikrein gene delivery on blood pressure reduction in spontaneously hypertensive rats (SHR). The human tissue kallikrein gene or cDNA was placed under the control of following promoters: the metallothionein gene metal response-element (MRE-pHK), albumin gene (ALB-pHK), Rous sarcoma virus 3' long terminal repeat (LTR) (RSV-cHK), and cytomegalovirus (CMV-cHK). A single injection of these kallikrein DNAs results in a significant reduction of blood pressure in SHR, which lasts for 5-6 weeks. Systemic delivery of CMV-cHK, RSV-cHK, and MRE-pHK has a greater effect on blood pressure reduction than ALB-pHK, whereas intraportal vein gene delivery of ALB-pHK is more effective than the other kallikrein DNA constructs. The degree of blood pressure reduction depends on the amount of administered DNA and the age of the animals. Reduction of blood pressure was observed in adult, but not young, SHR. The expression of human tissue kallikrein in rats was identified by an ELISA that is specific for human tissue kallikrein. No antibodies to either human tissue kallikrein or its DNA were detected in rat sera after somatic gene delivery. These results show that somatic gene delivery of human tissue kallikrein causes a lowering effect of systolic blood pressure in genetically hypertensive rats and provide valuable information for kallikrein gene therapy in the treatment of hypertension.

The roles of miRNAs in the onset of hearing and deafness are beginning to be revealed. Although there has been no reported link between chloride intracellular channel 5 (CLIC5) and the miR-183 family to date, we here present evidence that they are co-expressed in the inner ear and have functions that are related to stereocilia. Moreover, CLIC5 contains a single predicted and highly conserved miR-96/-182 binding site within its 3'-UTR. Our current results further show that miR-96/-182 and CLIC5 are co-expressed in HEI-OC1 cells, in which two isoforms of the CLIC5 protein exist. Furthermore, miR-96 and miR-182 were found to be specifically overexpressed in HEI-OC1 cells into which mimics of these molecules had been transfected by liposomes causing the downregulation of CLIC5 at both the mRNA and protein levels. Finally, miR-96/-182 specifically downregulate the expression of the luciferase reporter gene which was cloned into a mouse CLIC5 3'-UTR fragment containing the wild-type miR-96/-182 target sequence. Our findings thus suggest that CLIC5 is directly regulated by miR-96 and miR-182 and that the target sequence in this regard is located between nucleotides 760-766 within the CLIC5 3'-UTR.

Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-α2-3-galactose [Siaα2-3Gal] linkages on sialyloligosaccharides)- and human (Siaα2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.

The bradykinln B2 receptor (BDKRB2) has high affinity for the intact kinins, which mediate a wide spectrum of biological effects, including pain, inflammation, vasodilation, and smooth mnscle contraction and relaxation. In the present study, we have cloned and sequenced the gene encoding human bradyklnin B2 receptor from a human genomic library. The B2 receptor gene contains three exons separated by two introns. The first and second exons are noncoding, while the third exon contains the full-length coding region, which encodes a protein of 364 amino acids forming 7 transmembrane domains. The human B2 gene shares high sequence identity with rat and mouse B2 receptor genes and significant similarity with the gene encoding the angiotensin 11 type I receptor in the nucleotide sequence and exon-intron arrangement. In the 5′ flanking region, a consensus TATA box and several putative transcription factor-binding sites have been identified. Genomic Southern blot analysis showed that the B2 receptor is encoded by a single-copy gene that was localized to chromosome 14q32 by in situ hybridization. In a Southern blot analysis following reverse transcription and polymerase chain reaction, the human B2 receptor was found to be expressed in most human tissues.

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats., Author Summary Influenza epidemics exact a great toll on world health. Thus research to identify new anti-influenza virus strategies would be useful. Each of our cells contains antiviral factors that work to inhibit infection. A large component of this antiviral program is regulated by the interferon family of signaling molecules. Here, we seek to better understand how one of these antiviral factors, IFITM3, contributes to both baseline, as well as interferon-induced, antagonism of influenza A viral infection. We found that interferon prevents influenza A virus from entering our cells by blocking the virus' fusion with the cellular membrane. Furthermore, we learned that IFITM3 is required for this antiviral action of interferon, and that high levels of IFITM3 alone can produce a similar viral inhibition. Together, these results improve our understanding of how IFITM3 serves to defend us against viral invasion at a very early stage of infection.

Acquisition of α2-6 sialoside receptor specificity by α2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding α2-6 sialosides, we identified four variant viruses with amino acid substitutions in the hemagglutinin (S227N, D187G, E190G, and Q196R) that revealed modestly increased α2-6 and minimally decreased α2-3 binding by glycan array analysis. However, a mutant virus combining Q196R with mutations from previous pandemic viruses (Q226L and G228S) revealed predominantly α2-6 binding. Unlike the wild type H5N1, this mutant virus was transmitted by direct contact in the ferret model although not by airborne respiratory droplets. However, a reassortant virus with the mutant hemagglutinin, a human N2 neuraminidase and internal genes from an H5N1 virus was partially transmitted via respiratory droplets. The complex changes required for airborne transmissibility in ferrets suggest that extensive evolution is needed for H5N1 transmissibility in humans.