Increasing antiviral activity of surfactant protein d trimers by introducing residues from bovine serum collectins: dissociation of mannan-binding and antiviral activity.

Collectins contribute to host defence through interactions with glycoconjugates on pathogen surfaces. We have prepared recombinant trimeric neck and carbohydrate recognition domains (NCRD) of collectins, and we now show that the NCRD of bovine conglutinin and CL-46 (like that of CL-43) have greater intrinsic antiviral activity for influenza A virus (IAV) than the human SP-D NCRD (hSP-D-NCRD). The three serum collectins differ from SP-D by having insertions adjacent to amino acid 325 and substitution of hydrophobic residues for arginine 343. We previously showed that a three amino acid (RAK) insertion, as found in CL-43, increases antiviral activity and mannan-binding activity of the hSP-D-NCRD, while the substitution of valine at 343, as in conglutinin, more strongly increased these activities. Mannan-binding activity of collectins has been considered to predict for ability to bind to high mannose glycans on viruses or other pathogens. We now show, however, that combined mutants containing the RAK insertion and R343V or R343I substitutions have greatly increased mannan-binding ability, but lower IAV binding or inhibiting activity than mutants containing R343V or R343I substitutions only. These findings indicate differences in the recognition of glycan structures of mannan and IAV by the NCRD and emphasize the importance of the flanking sequences in determining the differing interactions of human SP-D and bovine serum collectins with mannose-rich glycoconjugates on IAV and other pathogens. Of interest, we show conservation of some monoclonal antibody-binding epitopes between bovine collectin NCRD and hSP-D, suggesting shared structural motifs.

Multiple components contribute to ability of saliva to inhibit influenza viruses.

INTRODUCTION: Saliva is a potentially important barrier against respiratory viral infection but its mechanism of action is not well studied. METHODS: We tested the antiviral activities of whole saliva, specific salivary gland secretions, and purified salivary proteins against strains of influenza A virus (IAV) in vitro. RESULTS: Whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibited IAV based on hemagglutination inhibition and neutralization assays. This differs from human immunodeficiency virus (HIV), for which only submandibular/sublingual secretions are reported to be inhibitory. Among purified salivary proteins, MUC5B, scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), histatins, and human neutrophil defensins (HNPs) inhibited IAV at the concentrations present in whole saliva. In contrast, some abundant salivary proteins (acidic proline-rich proteins and amylase) had no activity, nor did several other less abundant salivary proteins with known activity against HIV (e.g. thrombospondin or serum leukocyte protease inhibitor). Whole saliva and MUC5B did not inhibit neuraminidase activity of IAV and viral neutralizing and aggregating activity of MUC5B was potentiated by the neuraminidase inhibitor oseltamivir. Hence, MUC5B inhibits IAV by presenting a sialic acid ligand for the viral hemagglutinin. The mechanism of action of histatins requires further study. CONCLUSIONS: These findings indicate that saliva represents an important initial barrier to IAV infection and underline the complexity of host defense activity of oral secretions. Of interest, antiviral activity of saliva against IAV and HIV differs in terms of specific glandular secretions and proteins that are inhibitory.

Inhibition of influenza viral neuraminidase activity by collectins.

The collectins, lung surfactant proteins A and D (SP-A and SP-D), contribute to innate host defense against influenza A virus (IAV) in vivo. Although collectins bind to the viral hemagglutinin (HA) and inhibit early stages of viral infection in vitro, they also bind to the neuraminidase (NA) and inhibit NA activity. We used a variety of NA functional assays, viral strains and recombinant (mutant or wild type) collectins to characterize the mechanism of NA inhibition. NA inhibition by SP-D correlates with binding of its carbohydrate recognition domain (CRD) to oligomannose oligosaccharides on the viral hemagglutinin (HA). The effects of SP-D are additive with oseltamivir, consistent with differences in mechanism of action. NA inhibition was observed using fetuin or MDCK cells as a substrate, but not in assays using a soluble sialic acid analogue. Collectin multimerization and CRD binding properties are key determinants for NA inhibition. SP-D had greater NA inhibitory activity than mannose-binding lectin, which in turn had greater activity than SP-A. The markedly greater NA inhibitory activity of SP-D compared to SP-A may partly account for the finding that deletion of the SP-D gene in mice has a greater effect on viral replication in vivo.

Influenza a viruses upregulate neutrophil toll-like receptor 2 expression and function.

Neutrophils are involved in the initial host response to influenza A virus (IAV) infection and exhibit both activation and depressed function after exposure to the virus. We demonstrate that IAV causes rapid upregulation of Toll-like receptor 2 (TLR2) expression on neutrophils. The neutrophil agonists, formyl-methylpleucyl-alanine (fMLP), C5a and lipopolysaccharide did not alter neutrophil TLR2 expression, whereas PMA and the microbial TLR2 ligands, peptidoglycan (PGN) and zymosan, reduced it. To determine the functional significance of IAV-induced increase in TLR2 expression, IAV-treated neutrophils were exposed to PGN, Staphylococcus aureus (S. aureus) and zymosan. Pretreatment with IAV resulted in significantly increased uptake of S. aureus and zymosan and accelerated neutrophil apoptosis when combined with S. aureus. IAV-treated cells generated significantly more H(2)O(2) in response to PGN. These results indicate that IAV increases neutrophil surface expression of TLR2 and modulates functional responses to ligands that bind TLR2. These findings may clarify IAV-induced perturbation of neutrophil functions in vivo.

Acquired disorders of phagocyte function complicating medical and surgical illnesses.

There is evidence that acquired dysfunction of neutrophils, monocytes, or macrophages is an important cause of infection in patients with diabetes mellitus, renal or hepatic failure, alcoholism, autoimmune diseases, influenza or human immunodeficiency virus infection, burns, and trauma. Distinguishable mechanisms of acquired phagocyte dysfunction include inhibitory effects of metabolic disturbances (e.g., hyperglycemia, uremia), chemical toxins (e.g., ethanol), viral proteins on phagocyte activation, and pathologic activation of phagocytes in the circulation (e.g., after hemodialysis, burns, or cardiopulmonary bypass). Although the burden of morbidity and mortality resulting from acquired phagocyte dysfunction appears to be vast, research in this area has been hampered by the complexity of the underlying illnesses and by limitations of laboratory assays and clinical study methodology. Given the advent of improved assays of phagocyte functions and treatments that can enhance these functions, there is a pressing need for more prospective studies of acquired phagocyte dysfunction.

Surfactant protein D enhances clearance of influenza A virus from the lung in vivo.

Mice lacking surfactant protein surfactant protein D (SP-D(-/-)) and wild-type mice (SP-D(+/+)) were infected with influenza A virus (IAV) by intranasal instillation. IAV infection increased the endogenous SP-D concentration in wild-type mice. SP-D-deficient mice showed decreased viral clearance of the Phil/82 strain of IAV and increased production of inflammatory cytokines in response to viral challenge. However, the less glycosylated strain of IAV, Mem/71, which is relatively resistant to SP-D in vitro, was cleared efficiently from the lungs of SP-D(-/-) mice. Viral clearance of the Phil/82 strain of IAV and the cytokine response were both normalized by the coadministration of recombinant SP-D. Since the airway is the usual portal of entry for influenza A virus and other respiratory pathogens, SP-D is likely to play an important role in innate defense responses to IAV.

Increased antiviral and opsonic activity of a highly multimerized collectin chimera.

Altering the carbohydrate binding properties of surfactant protein D (SP-D) [e.g., by replacing its carbohydrate recognition domain (CRD) with that of either mannose binding lectin (MBL) or conglutinin] can increase its activity against influenza A virus (IAV). The current study demonstrates that the degree of multimerization of SP-D is another independent determinant of antiviral activity. A chimeric collectin containing the N-terminus and collagen domain of human SP-D and the CRD of MBL formed high-molecular-weight multimers similar to those previously described for human SP-D. Using several complementary assays, and diverse viral strains, the chimeric multimers showed greater anti-IAV activity than similarly multimerized preparations of SP-D or incompletely oligomerized preparations of the chimera. More highly multimerized preparations of the chimera also caused greater increases in uptake of IAV by neutrophils. These studies may have implications for development of collectins as therapeutic agents and understanding of natural variations in susceptibility to IAV infection.

Prophylaxis and treatment of influenza virus infection.

Influenza virus infections remain an important cause of morbidity and mortality. Furthermore, a recurrence of pandemic influenza remains a real possibility. There are now effective ways to both prevent and treat influenza. Prevention of infection is most effectively accomplished by vaccination. Vaccination with the inactivated, intramuscular influenza vaccine has been clearly demonstrated to reduce serious morbidity and mortality associated with influenza infection, especially in groups of patients at high risk (e.g. the elderly). However, the inactivated, intramuscular vaccine does not strongly induce cell-mediated or mucosal immune responses, and protection induced by the vaccine is highly strain specific. Live, attenuated influenza vaccines administered intranasally have been studied in clinical trials and shown to elicit stronger mucosal and cell-mediated immune responses. Live, attenuated vaccines appear to be more effective for inducing protective immunity in children or the elderly than inactivated, intramuscular vaccines. Additionally, novel vaccine methodologies employing conserved components of influenza virus or viral DNA are being developed. Preclinical studies suggest that these approaches may lead to methods of vaccination that could induce immunity against diverse strains or subtypes of influenza. Because of the limitations of vaccination, antiviral therapy continues to play an important role in the control of influenza. Two major classes of antivirals have demonstrated ability to prevent or treat influenza in clinical trials: the adamantanes and the neuraminidase inhibitors. The adamantanes (amantadine and rimantadine) have been in use for many years. They inhibit viral uncoating by blocking the proton channel activity of the influenza A viral M2 protein. Limitations of the adamantanes include lack of activity against influenza B, toxicity (especially in the elderly), and the rapid development of resistance. The neuraminidase inhibitors were designed to interfere with the conserved sialic acid binding site of the viral neuraminidase and act against both influenza A and B with a high degree of specificity when administered by the oral (oseltamivir) or inhaled (zanamivir) route. The neuraminidase inhibitors have relatively low toxicity, and viral resistance to these inhibitors appears to be uncommon. Additional novel antivirals that target other phases of the life cycle of influenza are in preclinical development. For example, recombinant collectins inhibit replication of influenza by binding to the viral haemagglutinin as well as altering phagocyte responses to the virus. Recombinant techniques have been used for generation of antiviral proteins (e.g. modified collectins) or oligonucleotides. Greater understanding of the biology of influenza viruses has already resulted in significant advances in the management of this important pathogen. Further advances in vaccination and antiviral therapy of influenza should remain a high priority.

Neutrophil survival is markedly reduced by incubation with influenza virus and Streptococcus pneumoniae: role of respiratory burst.

Bacterial superinfections are an important cause of morbidity and mortality during influenza A virus (IAV) epidemics. We demonstrate that incubation with the combination of IAV and Streptococcus pneumoniae caused marked reductions in survival of neutrophils in vitro compared with treatment with control buffer or IAV or S. pneumoniae alone. This cooperative effect was in part mediated by acceleration of neutrophil apoptosis as evidenced by increases in annexin-V binding and caspase-3 activation. However, GM-CSF did not increase survival of neutrophils exposed to IAV and S. pneumoniae. IAV enhanced neutrophil uptake of S. pneumoniae significantly. Furthermore, the combination of IAV and S. pneumoniae caused significantly more hydrogen peroxide production than IAV or S. pneumoniae alone. This increased respiratory burst activity contributed to the diminished neutrophil survival caused by IAV and S. pneumoniae. The NADPH oxidase inhibitor, diphenyleneiodonium, significantly improved survival of neutrophils treated with IAV and S. pneumoniae. These findings may help to explain the increased susceptibility of IAV-infected patients to infections with S. pneumoniae.

Brief report: improvements in the behavior of children with autism following massage therapy.

Twenty children with autism, ages 3 to 6 years, were randomly assigned to massage therapy and reading attention control groups. Parents in the massage therapy group were trained by a massage therapist to massage their children for 15 minutes prior to bedtime every night for 1 month and the parents of the attention control group read Dr. Seuss stories to their children on the same time schedule. Conners Teacher and Parent scales, classroom and playground observations, and sleep diaries were used to assess the effects of therapy on various behaviors, including hyperactivity, stereotypical and off-task behavior, and sleep problems. Results suggested that the children in the massage group exhibited less stereotypic behavior and showed more on-task and social relatedness behavior during play observations at school, and they experienced fewer sleep problems at home.

Mechanism of binding of surfactant protein D to influenza A viruses: importance of binding to haemagglutinin to antiviral activity.

Collectins are important in the initial containment of a variety of pathogens, including influenza A virus (IAV). We provide the first systematic evaluation of the oligosaccharide-binding sites for pulmonary surfactant protein D (SP-D) on specific IAV coat glycoproteins and define the relationship between this binding and antiviral activity. With the use of several techniques, SP-D was found to bind via its carbohydrate-recognition domain (CRD) to mannosylated, N-linked carbohydrates on the HA(1) domain of the haemagglutinin (HA) and on the neuraminidase of IAV. Using a set of IAV strains that differed in the level and site of glycosylation, and a panel of recombinant collectins, we found that binding of SP-D to the globular domain of the HA was critical in mediating the inhibition of viral haemagglutination activity and infectivity. We also demonstrated that the pattern of binding of a collectin to IAV glycoproteins can be modified by altering the monosaccharide-binding affinity of its CRD or by linking the CRD to a different N-terminal/collagen domain. These studies clarify the mechanisms of viral neutralization by collectins and might be useful in engineering collectins for enhanced antiviral activity.

Enhanced antiviral and opsonic activity of a human mannose-binding lectin and surfactant protein D chimera.

The carbohydrate recognition domains (CRDs) of human serum mannose-binding lectin (MBL) and pulmonary surfactant protein D (SP-D) have distinctive monosaccharide-binding properties, and their N-terminal and collagen domains have very different quaternary structures. We produced a chimeric protein containing the N terminus and collagen domain of human SP-D and the neck region and CRD of human MBL (SP-D/MBLneck+CRD) to create a novel human collectin. The chimera bound to influenza A virus (IAV), inhibited IAV hemagglutination activity and infectivity, and induced aggregation of viral particles to a much greater extent than MBL. Furthermore, SP-D/MBLneck+CRD caused much greater increases in neutrophil uptake of, and respiratory burst responses to, IAV than MBL. These results indicate that pathogen interactions mediated by the MBL CRD are strongly influenced by the N-terminal and collagen-domain backbone to which it is attached. The presence of the CRD of MBL in the chimera resulted in altered monosaccharide binding properties compared with SP-D. As a result, the chimera caused greater aggregation and neutralization of IAV than SP-D. Distinctive functional properties of collectin collagenous domains and CRDs can be exploited to generate novel human collectins with potential for therapy of influenza.

Circulating neutrophil CD18 receptor expression and ischemic flap neutrophil infiltration in a guinea pig model.

This article demonstrates a correlation between circulating neutrophil CD18 expression, neutrophil infiltration, and varying periods of ischemia induced in guinea pig island skin flaps. Fifty adult female Hartley guinea pigs were equally separated into a control group, a sham group, and ischemic groups of 2, 4, and 10 hours. All, except those in the control group, had single guinea pig island flank skin flaps raised. Systemic neutrophil surface receptor (CD18) expression was analyzed with monoclonal antibodies, and flap skin biopsy specimens were analyzed for neutrophil infiltration. The results show that neutrophil counts and receptor detection increase as flap ischemia increases. However, a trend toward declining receptor expression was observed in the 10-hour ischemic group. In conclusion, systemic neutrophil adhesion receptor upregulation is correlated with cutaneous flap neutrophil infiltration and ischemia-reperfusion injury in a guinea pig model. A trend toward declining receptor expression with advanced ischemia was observed.

Collectins and pulmonary innate immunity.

The surfactant-associated proteins SP-A and SP-D are members of a family of host defense lectins, designated collectins. There is increasing evidence that these pulmonary, epithelial-derived proteins are important components of the innate immune response to microbial challenge and participate in other aspects of immune and inflammatory regulation within the lung. Both proteins bind to glycoconjugates and/or lipid moieties expressed by a wide variety of microorganisms, and to certain organic particles, such as pollens. SP-A and SP-D have the capacity to modulate leukocyte function and, in some circumstances; to opsonize and enhance the killing of microorganisms. The biologic activity of cell wall components, such as Gram-negative bacterial polysaccharides, or viral glycoproteins, such as the hemagglutinin of influenza viruses, may be altered by interactions with collectins. In addition, complementary or cooperative interactions between SP-A, SP-D and other host defense lectins could contribute to the efficiency of this defense system. Collectins could play particularly important roles in settings of inadequate or impaired specific immunity, and acquired alterations in the levels of active collectins within the airspaces and distal airways may increase susceptibility to infection.

Enhanced anti-influenza activity of a surfactant protein D and serum conglutinin fusion protein.

We previously demonstrated that bovine serum conglutinin has markedly greater ability to inhibit influenza A virus (IAV) infectivity than other collectins. We now show that recombinant conglutinin and a chimeric protein containing the NH(2) terminus and collagen domain of rat pulmonary surfactant protein D (rSP-D) fused to the neck region and carbohydrate recognition domain (CRD) of conglutinin (termed SP-D/Cong(neck+CRD)) have markedly greater ability to inhibit infectivity of IAV than wild-type recombinant rSP-D, confirming that the potent IAV-neutralizing activity of conglutinin resides in its neck region and CRD. Furthermore, by virtue of incorporation of the NH(2) terminus and collagen domain of SP-D, SP-D/Cong(neck+CRD) caused substantially greater aggregation of IAV particles and enhancement of neutrophil binding of, and H(2)O(2) responses to, IAV than recombinant conglutinin or recombinant rSP-D. Hence, SP-D/Cong(neck+CRD) combined favorable antiviral and opsonic properties of conglutinin and SP-D. This study demonstrates an association of specific structural domains of SP-D and conglutinin with specific functional properties and illustrates that antimicrobial activities of wild-type collectins can be enhanced through recombinant strategies.

Long-term maintenance of infant memory.

The present experiments with human infants asked whether periodic nonverbal reminders could maintain a memory established at 2 months of age over a substantial period of development. In Experiment 1, a reactivation reminder recovered infants' forgotten memory after 3 weeks, but a reinstatement reminder did not. In Experiment 2, 2-month-olds received a reminder every 3 weeks through 6(1/2) months of age and a final test at 7(1/4) months of age. A preliminary retention test preceded each reminder; which type of reminder (reinstatement or reactivation) infants received depended on performance during this test. Infants exhibited significant retention 4(1/2) months later, and most remembered 5(1/4) months later, when infants outgrew the task. Untrained controls exhibited no retention after any delay. These data confirm that periodic reminders can maintain early memories over significant periods of development and challenge popular claims that preverbal human infants cannot maintain memories over the long term because of neural immaturity or an inability to rehearse experiences by talking about them.

Influenza A virus up-regulates neutrophil adhesion molecules and adhesion to biological surfaces.

Influenza A virus (IAV) binds to sialylated neutrophil surface components (e.g., CD43 and sialyl Lewisx antigen) and induces both activation and functional depression of neutrophils. We report that IAV enhanced neutrophil adhesion to surfaces coated with serum or serum components, but not to uncoated plastic. IAV up-regulated expression of integrins (CD11b and CD11c) and carcinoembryonic-related antigens on neutrophils, while reducing expression of CD43, L-selectin, and P-selectin glycoprotein ligand (PSGL). Although treatment of neutrophils with elastase or Osialoglycoprotease decreased surface CD43 and PSGL, they did not reduce binding of IAV to neutrophils, implying that IAV can bind to alternate binding sites in the absence of CD43. Pretreatment of neutrophils with elastase also did not prevent IAV from increasing expression of integrins and enhancing adhesion. Up-regulation of adhesion molecules and adhesion are important, previously unrecognized, features of neutrophil activation by IAV. Further studies will be needed to clarify the mechanism of these effects.