Influenza Transmission in the Mother-Infant Dyad Leads to Severe Disease, Mammary Gland Infection, and Pathogenesis by Regulating Host Responses.

Seasonal influenza viruses are typically restricted to the human upper respiratory tract whereas influenza viruses with greater pathogenic potential often also target extra-pulmonary organs. Infants, pregnant women, and breastfeeding mothers are highly susceptible to severe respiratory disease following influenza virus infection but the mechanisms of disease severity in the mother-infant dyad are poorly understood. Here we investigated 2009 H1N1 influenza virus infection and transmission in breastfeeding mothers and infants utilizing our developed infant-mother ferret influenza model. Infants acquired severe disease and mortality following infection. Transmission of the virus from infants to mother ferrets led to infection in the lungs and mother mortality. Live virus was also found in mammary gland tissue and expressed milk of the mothers which eventually led to milk cessation. Histopathology showed destruction of acini glandular architecture with the absence of milk. The virus was localized in mammary epithelial cells of positive glands. To understand the molecular mechanisms of mammary gland infection, we performed global transcript analysis which showed downregulation of milk production genes such as Prolactin and increased breast involution pathways indicated by a STAT5 to STAT3 signaling shift. Genes associated with cancer development were also significantly increased including JUN, FOS and M2 macrophage markers. Immune responses within the mammary gland were characterized by decreased lymphocyte-associated genes CD3e, IL2Ra, CD4 with IL1ß upregulation. Direct inoculation of H1N1 into the mammary gland led to infant respiratory infection and infant mortality suggesting the influenza virus was able to replicate in mammary tissue and transmission is possible through breastfeeding. In vitro infection studies with human breast cells showed susceptibility to H1N1 virus infection. Together, we have shown that the host-pathogen interactions of influenza virus infection in the mother-infant dyad initiate immunological and oncogenic signaling cascades within the mammary gland. These findings suggest the mammary gland may have a greater role in infection and immunity than previously thought.

A New Multi-Mode Perfusion System for Ex Vivo Heart Perfusion Study.

Ex vivo heart perfusion has been shown to be an effective means of facilitating the resuscitation and assessment of donor hearts for cardiac transplantation. Over the last ten years however, only a few ex vivo perfusion systems have been developed for this application. While results have been promising, a system capable of facilitating multiple perfusion strategies on the same platform has not yet been realized. In this paper, the design, development and testing of a novel and modular ex vivo perfusion system is described. The system is capable of operating in three unique primary modes: the traditional Langendorff Mode, Pump-Supported Working-Mode, and Passive Afterload Working-Mode. In each mode, physiological hemodynamic parameters can be produced by managing perfusion settings. To evaluate heart viability, six experiments were conducted using porcine hearts and measuring several parameters including: pH, aortic pressure, lactate metabolism, coronary vascular resistance (CVR), and myocardial oxygen consumption. Pressure-volume relationship measurements were used to assess left ventricular contractility in each Working Mode. Hemodynamic and metabolic conditions remained stable and consistent across 4 h of ex vivo heart perfusion on the ex vivo perfusion system, validating the system as a viable platform for future development of novel preservation and assessment strategies.

Pathogenic influenza B virus in the ferret model establishes lower respiratory tract infection.

Influenza B viruses have become increasingly more prominent during influenza seasons. Influenza B infection is typically considered a mild disease and receives less attention than influenza A, but has been causing 20 to 50 % of the total influenza incidence in several regions around the world. Although there is increasing evidence of mid to lower respiratory tract diseases such as bronchitis and pneumonia in influenza B patients, little is known about the pathogenesis of recent influenza B viruses. Here we investigated the clinical and pathological profiles of infection with strains representing the two current co-circulating B lineages (B/Yamagata and B/Victoria) in the ferret model. Specifically, we studied two B/Victoria (B/Brisbane/60/2008 and B/Bolivia/1526/2010) and two B/Yamagata (B/Florida/04/2006 and B/Wisconsin/01/2010) strain infections in ferrets and observed strain-specific but not lineage-specific pathogenicity. We found B/Brisbane/60/2008 caused the most severe clinical illness and B/Brisbane/60/2008 and the B/Yamagata strains instigated pathology in the middle to lower respiratory tract. Importantly, B/Brisbane/60/2008 established efficient lower respiratory tract infection with high viral burden. Our phylogenetic analyses demonstrate profound reassortment among recent influenza B viruses, which indicates the genetic make-up of B/Brisbane/60/2008 differs from the other strains. This may explain the pathogenicity difference post-infection in ferrets.

Sequencing, annotation, and characterization of the influenza ferret infectome.

Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.

Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones.

The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic ß-cells, which leads to inactivation of the ß-cell proliferating activity of Tmem27. This role of BACE2 in the control of ß-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme ß-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.

Seasonal H1N1 influenza virus infection induces cross-protective pandemic H1N1 virus immunity through a CD8-independent, B cell-dependent mechanism.

During the 2009 H1N1 influenza virus pandemic (pdmH1N1) outbreak, it was found that most individuals lacked antibodies against the new pdmH1N1 virus, and only the elderly showed anti-hemagglutinin (anti-HA) antibodies that were cross-reactive with the new strains. Different studies have demonstrated that prior contact with the virus can confer protection against strains with some degree of dissimilarity; however, this has not been sufficiently explored within the context of a pdmH1N1 virus infection. In this study, we have found that a first infection with the A/Brisbane/59/2007 virus strain confers heterologous protection in ferrets and mice against a subsequent pdmH1N1 (A/Mexico/4108/2009) virus infection through a cross-reactive but non-neutralizing antibody mechanism. Heterologous immunity is abrogated in B cell-deficient mice but maintained in CD8(-/-) and perforin-1(-/-) mice. We identified cross-reactive antibodies from A/Brisbane/59/2007 sera that recognize non-HA epitopes in pdmH1N1 virus. Passive serum transfer showed that cross-reactive sH1N1-induced antibodies conferred protection in naive recipient mice during pdmH1N1 virus challenge. The presence or absence of anti-HA antibodies, therefore, is not the sole indicator of the effectiveness of protective cross-reactive antibody immunity. Measurement of additional antibody repertoires targeting the non-HA antigens of influenza virus should be taken into consideration in assessing protection and immunization strategies. We propose that preexisting cross-protective non-HA antibody immunity may have had an overall protective effect during the 2009 pdmH1N1 outbreak, thereby reducing disease severity in human infections.

Differential pathological and immune responses in newly weaned ferrets are associated with a mild clinical outcome of pandemic 2009 H1N1 infection.

Young children are typically considered a high-risk group for disease associated with influenza virus infection. Interestingly, recent clinical reports suggested that young children were the smallest group of cases with severe pandemic 2009 H1N1 (H1N1pdm) influenza virus infection. Here we established a newly weaned ferret model for the investigation of H1N1pdm infection in young age groups compared to adults. We found that young ferrets had a significantly milder fever and less weight loss than adult ferrets, which paralleled the mild clinical symptoms in the younger humans. Although there was no significant difference in viral clearance, disease severity was associated with pulmonary pathology, where newly weaned ferrets had an earlier pathology improvement. We examined the immune responses associated with protection of the young age group during H1N1pdm infection. We found that interferon and regulatory interleukin-10 responses were more robust in the lungs of young ferrets. In contrast, myeloperoxidase and major histocompatibility complex responses were persistently higher in the adult lungs; as well, the numbers of inflammation-prone granulocytes were highly elevated in the adult peripheral blood. Importantly, we observed that H1N1pdm infection triggered formation of lung structures that resembled inducible bronchus-associated lymphoid tissues (iBALTs) in young ferrets which were associated with high levels of homeostatic chemokines CCL19 and CXCL13, but these were not seen in the adult ferrets with severe disease. These results may be extrapolated to a model of the mild disease seen in human children. Furthermore, these mechanistic analyses provide significant new insight into the developing immune system and effective strategies for intervention and vaccination against respiratory viruses.

BACE1 inhibitors: a head group scan on a series of amides.

A series of amides bearing a variety of amidine head groups was investigated as BACE1 inhibitors with respect to inhibitory activity in a BACE1 enzyme as well as a cell-based assay. Determination of their basicity as well as their properties as substrates of P-glycoprotein revealed that a 2-amino-1,3-oxazine head group would be a suitable starting point for further development of brain penetrating compounds for potential Alzheimer's disease treatment.

CXCL10 contributes to p38-mediated apoptosis in primary T lymphocytes in vitro.

CXCL10 is part of the group of interferon-stimulated genes and it plays an important role during different viral infections by inducing cell activation, chemotaxis and lymphocyte priming toward the Th1 phenotype. In this study, we investigated in vitro the effects of CXCL10 in activated human primary T lymphocytes in terms of apoptosis or survival, and delineated the signaling pathways that are involved. CXCL10, in combination with IL-2 and/or IFNα, induces apoptosis in T lymphocytes. Moreover, CXCL10-induced activation of CXCR3 also triggers pro-survival signals that can be blocked by pertussis toxin. The analysis of the downstream signaling kinases shows that apoptosis is p38 MAPK-dependent and the pro-survival signals rely on the sustained activation of PI3K and the transient activation of Akt. On the other hand, the transient activation of p44/p42 ERK did not have an impact on T lymphocyte survival. We propose an immunological model in which CXCL10, together with other co-stimulating cytokines, participates in the activation of T lymphocytes, promotes survival and expansion of certain lymphocyte subsets, and induces chemotaxis toward the infected tissues. On the other hand, CXCL10 might contribute to the triggering of apoptosis in other subsets of T lymphocytes, including those lymphocytes that were transiently activated but later lacked the appropriate sets of specific co-stimulating signals to ensure their survival.

Molecular recognition at the active site of factor Xa: cation-π interactions, stacking on planar peptide surfaces, and replacement of structural water.

Factor Xa, a serine protease from the blood coagulation cascade, is an ideal enzyme for molecular recognition studies, as its active site is highly shape-persistent and features distinct, concave sub-pockets. We developed a family of non-peptidic, small-molecule inhibitors with a central tricyclic core orienting a neutral heterocyclic substituent into the S1 pocket and a quaternary ammonium ion into the aromatic box in the S4 pocket. The substituents were systematically varied to investigate cation-π interactions in the S4 pocket, optimal heterocyclic stacking on the flat peptide walls lining the S1 pocket, and potential water replacements in both the S1 and the S4 pockets. Structure-activity relationships were established to reveal and quantify contributions to the binding free enthalpy, resulting from single-atom replacements or positional changes in the ligands. A series of high-affinity ligands with inhibitory constants down to K(i)=2 nM were obtained and their proposed binding geometries confirmed by X-ray co-crystal structures of protein-ligand complexes.

Molecular characterization of in vivo adjuvant activity in ferrets vaccinated against influenza virus.

The 2009 H1N1 influenza pandemic has prompted a significant need for the development of efficient, single-dose, adjuvanted vaccines. Here we investigated the adjuvant potential of CpG oligodeoxynucleotide (ODN) when used with a human seasonal influenza virus vaccine in ferrets. We found that the CpG ODN-adjuvanted vaccine effectively increased antibody production and activated type I interferon (IFN) responses compared to vaccine alone. Based on these findings, pegylated IFN-alpha2b (PEG-IFN) was also evaluated as an adjuvant in comparison to CpG ODN and complete Freund's adjuvant (CFA). Our results showed that all three vaccines with adjuvant added prevented seasonal human A/Brisbane/59/2007 (H1N1) virus replication more effectively than did vaccine alone. Gene expression profiles indicated that, as well as upregulating IFN-stimulated genes (ISGs), CpG ODN enhanced B-cell activation and increased Toll-like receptor 4 (TLR4) and IFN regulatory factor 4 (IRF4) expression, whereas PEG-IFN augmented adaptive immunity by inducing major histocompatibility complex (MHC) transcription and Ras signaling. In contrast, the use of CFA as an adjuvant induced limited ISG expression but increased the transcription of MHC, cell adhesion molecules, and B-cell activation markers. Taken together, our results better characterize the specific molecular pathways leading to adjuvant activity in different adjuvant-mediated influenza virus vaccinations.

Direct association between pharyngeal viral secretion and host cytokine response in severe pandemic influenza.

BACKGROUND: Severe disease caused by 2009 pandemic influenza A/H1N1virus is characterized by the presence of hypercytokinemia. The origin of the exacerbated cytokine response is unclear. As observed previously, uncontrolled influenza virus replication could strongly influence cytokine production. The objective of the present study was to evaluate the relationship between host cytokine responses and viral levels in pandemic influenza critically ill patients. METHODS: Twenty three patients admitted to the ICU with primary viral pneumonia were included in this study. A quantitative PCR based method targeting the M1 influenza gene was developed to quantify pharyngeal viral load. In addition, by using a multiplex based assay, we systematically evaluated host cytokine responses to the viral infection at admission to the ICU. Correlation studies between cytokine levels and viral load were done by calculating the Spearman correlation coefficient. RESULTS: Fifteen patients needed of intubation and ventilation, while eight did not need of mechanical ventilation during ICU hospitalization. Viral load in pharyngeal swabs was 300 fold higher in the group of patients with the worst respiratory condition at admission to the ICU. Pharyngeal viral load directly correlated with plasma levels of the pro-inflammatory cytokines IL-6, IL-12p70, IFN-γ, the chemotactic factors MIP-1ß, GM-CSF, the angiogenic mediator VEGF and also of the immuno-modulatory cytokine IL-1ra (p < 0.05). Correlation studies demonstrated also the existence of a significant positive association between the levels of these mediators, evidencing that they are simultaneously regulated in response to the virus. CONCLUSIONS: Severe respiratory disease caused by the 2009 pandemic influenza virus is characterized by the existence of a direct association between viral replication and host cytokine response, revealing a potential pathogenic link with the severe disease caused by other influenza subtypes such as H5N1.

In vivo ribavirin activity against severe pandemic H1N1 Influenza A/Mexico/4108/2009.

The use of ribavirin in influenza treatment is a matter of debate. Due to adamantine- and oseltamivir-resistant strains of the current pandemic H1N1 (pdmH1N1) influenza viruses, the demand for alternative antiviral treatments has increased. This study demonstrated the potent antiviral effects of ribavirin in a mouse model of pdmH1N1 influenza infection (A/Mexico/4108/2009). It was found that treatment with 40 mg ribavirin kg⁻¹ day⁻¹ partially protected the animals if initiated immediately upon infection. Administration of similar concentrations on subsequent days or immediate therapy with lower doses efficiently delayed disease progression. Correlation studies showed a direct relationship between low viral titres in the lung during the early stages of infection with animal survival in ribavirin-treated animals. Reduced lung pathology in animals treated with ribavirin following infection also indicated the importance of immediate treatment. This study revealed the antiviral properties of ribavirin and these results justify comprehensive clinical studies for the use of ribavirin against influenza virus in future outbreaks.

Discovery of a factor Xa inhibitor (3R,4R)-1-(2,2-difluoro-ethyl)-pyrrolidine-3,4-dicarboxylic acid 3-[(5-chloro-pyridin-2-yl)-amide] 4-[[2-fluoro-4-(2-oxo-2H-pyridin-1-yl)-phenyl]-amide] as a clinical candidate.

A series of (3R,4R)-pyrrolidine-3,4-dicarboxylic acid amides was investigated with respect to their factor Xa inhibitory activity, selectivity, pharmacokinetic properties, and ex vivo antithrombotic activity. The clinical candidate from this series, R1663, exhibits excellent selectivity against a panel of serine proteases and good pharmacokinetic properties in rats and monkeys. A Phase I clinical study with R1663 has been finalized.

Host adaptive immunity deficiency in severe pandemic influenza.

INTRODUCTION: Pandemic A/H1N1/2009 influenza causes severe lower respiratory complications in rare cases. The association between host immune responses and clinical outcome in severe cases is unknown. METHODS: We utilized gene expression, cytokine profiles and generation of antibody responses following hospitalization in 19 critically ill patients with primary pandemic A/H1N1/2009 influenza pneumonia for identifying host immune responses associated with clinical outcome. Ingenuity pathway analysis 8.5 (IPA) (Ingenuity Systems, Redwood City, CA) was used to select, annotate and visualize genes by function and pathway (gene ontology). IPA analysis identified those canonical pathways differentially expressed (P < 0.05) between comparison groups. Hierarchical clustering of those genes differentially expressed between groups by IPA analysis was performed using BRB-Array Tools v.3.8.1. RESULTS: The majority of patients were characterized by the presence of comorbidities and the absence of immunosuppressive conditions. pH1N1 specific antibody production was observed around day 9 from disease onset and defined an early period of innate immune response and a late period of adaptive immune response to the virus. The most severe patients (n = 12) showed persistence of viral secretion. Seven of the most severe patients died. During the late phase, the most severe patient group had impaired expression of a number of genes participating in adaptive immune responses when compared to less severe patients. These genes were involved in antigen presentation, B-cell development, T-helper cell differentiation, CD28, granzyme B signaling, apoptosis and protein ubiquitination. Patients with the poorest outcomes were characterized by proinflammatory hypercytokinemia, along with elevated levels of immunosuppressory cytokines (interleukin (IL)-10 and IL-1ra) in serum. CONCLUSIONS: Our findings suggest an impaired development of adaptive immunity in the most severe cases of pandemic influenza, leading to an unremitting cycle of viral replication and innate cytokine-chemokine release. Interruption of this deleterious cycle may improve disease outcome.

Correlative ex situ and Liquid-Cell TEM Observation of Bacterial Cell Membrane Damage Induced by Rough Surface Topology.

BACKGROUND: Nanoscale surface roughness has been suggested to have antibacterial and antifouling properties. Several existing models have attempted to explain the antibacterial mechanism of nanoscale rough surfaces without direct observation. Here, conventional and liquid-cell TEM are implemented to observe nanoscale bacteria/surface roughness interaction. The visualization of such interactions enables the inference of possible antibacterial mechanisms. METHODS AND RESULTS: Nanotextures are synthesized on biocompatible polymer microparticles (MPs) via plasma etching. Both conventional and liquid-phase transmission electron microscopy observations suggest that these MPs may cause cell lysis via bacterial binding to a single protrusion of the nanotexture. The bacterium/protrusion interaction locally compromises the cell wall, thus causing bacterial death. This study suggests that local mechanical damage and leakage of the cytosol kill the bacteria first, with subsequent degradation of the cell envelope. CONCLUSION: Nanoscale surface roughness may act via a penetrative bactericidal mechanism. This insight suggests that future research may focus on optimizing bacterial binding to individual nanoscale projections in addition to stretching bacteria between nanopillars. Further, antibacterial nanotextures may find use in novel applications employing particles in addition to nanotextures on fibers or films.

Etoposide-Bound Magnetic Nanoparticles Designed for Remote Targeting of Cancer Cells Disseminated Within Cerebrospinal Fluid Pathways.

Magnetic nanoparticles (MNPs) have potential for enhancing drug delivery in selected cancer patients, including those which have cells that have disseminated within cerebrospinal fluid (CSF) pathways. Here, we present data related to the creation and in vitro use of new two-part MNPs consisting of magnetic gold-iron alloy cores which have streptavidin binding sites, and are coated with biotinylated etoposide. Etoposide was chosen due to its previous use in the CSF and ease of biotinylation. Etoposide magnetic nanoparticles ("Etop-MNPs") were characterized by several different methods, and moved at a distance by surface-walking of MNP clusters, which occurs in response to a rotating permanent magnet. Human cell lines including D283 (medulloblastoma), U138 (glioblastoma), and H2122 (lung adenocarcinoma) were treated with direct application of Etop-MNPs (and control particles), and after remote particle movement. Cell viability was determined by MTT assay and trypan blue exclusion. Results indicated that the biotinylated etoposide was successfully bound to the base MNPs, with the hybrid particle attaining a maximum velocity of 0.13 ± 0.018 cm/sec. Etop-MNPs killed cancer cells in a dose-dependent fashion, with 50 ± 6.8% cell killing of D283 cells (for example) with 24 h of treatment after remote targeting. U138 and H2122 cells were found to be even more susceptible to the killing effect of Etop-MNPs than D283 cells. These findings indicate that the novel Etop-MNPs have a cytotoxic effect, and can be moved relatively rapidly at physiologic distances, using a rotating magnet. While further testing is needed, intrathecal administration of Etop-MNPs holds promise for magnetically-enhanced eradication of cancer cells distributed within CSF pathways, particularly if given early in the course of the disease.

TEM Studies on Antibacterial Mechanisms of Black Phosphorous Nanosheets.

PURPOSE: Recently, two-dimensional (2D) nanomaterials are gaining tremendous attention as novel antibacterial platforms to combat against continuously evolving antimicrobial resistance levels. Among the family of 2D nanomaterials, black phosphorus (BP) nanosheets have demonstrated promising potential for biomedical applications. However, there is a need to gain nanoscale insights of the antibacterial activity of BP nanosheets which lies at the center of technical challenges. METHODS: Ultra-large BP nanosheets were synthesized by liquid-exfoliation method in the eco-friendly deoxygenated water. Synthesized BP nanosheets were characterized by TEM, AFM, and Raman spectroscopy techniques and their chemical stability was evaluated by EDS and EELS elemental analysis. The antibacterial activity of BP nanosheets was evaluated at nanoscale by the ultramicrotome TEM technique. Further, HAADF-STEM image and EDS elemental line map of the damaged bacterium were utilized to analyze the presence of diagnostic ions. Supportive SEM and ATR-FTIR studies were carried out to confirm the bacterial cell wall damage. In vitro colony counting method was utilized to evaluate the antibacterial performance of ultra-large BP nanosheets. RESULTS: Elemental EELS and EDS analysis of BP nanosheets stored in deoxygenated water confirmed the absence of oxygen peak. TEM studies indicate the various events of bacterial cell damage with the lost cellular metabolism and structural integrity. Colony counting test results show that as-synthesized BP nanosheets (100 µg/mL) can kill ~95% bacteria within 12 hours. CONCLUSION: TEM studies demonstrate the various events of E. coli membrane damage and the loss of structural integrity. These events include the BP nanosheets interaction with the bacterial cell wall, cytoplasmic leakage, detachment of cytoplasm from the cell membrane, reduced density of lipid bilayer and agglomerated DNA structure. The EDS elemental line mapping of the damaged bacterium confirms the disrupted cell membrane permeability and the lost cellular metabolism. SEM micrographs and ATR-FTIR supportive results confirm the bacterial cell wall damage.

Fragment-based screening using surface plasmon resonance technology.

Surface plasmon resonance (SPR) technology has emerged as a new and powerful technique to investigate the interaction between low-molecular-weight molecules and target proteins. In the present work, the authors assemble from a large compound collection a library of 2226 molecules (fragments having low molecular weights between 100 and 300 Da) to screen them for binding to chymase, a serine protease. Both the active chymase and a zymogen-like form of the protein were used in parallel to distinguish between specific and unspecific binding. The relative ligand-binding activity of the immobilized protein was periodically measured with a reference compound. The screening experiments were performed at 25 degrees C at a fragment concentration of 200 microM in the presence of 2% DMSO. Applying the filter cascade, affinity-selectivity-competition (competition with reference compounds and cross-competition with fragments), 80 compounds show up as positive screening hits. Competition experiments between fragments show that they bind to different parts of the active site. Of 36 fragments co-crystallized for X-ray studies, 12 could be located in the active site of the protein. These results validate the authors' library and demonstrate that the application of SPR technology as a filter in fragment screening can be achieved successfully.

Th1 and Th17 hypercytokinemia as early host response signature in severe pandemic influenza.

INTRODUCTION: Human host immune response following infection with the new variant of A/H1N1 pandemic influenza virus (nvH1N1) is poorly understood. We utilize here systemic cytokine and antibody levels in evaluating differences in early immune response in both mild and severe patients infected with nvH1N1. METHODS: We profiled 29 cytokines and chemokines and evaluated the haemagglutination inhibition activity as quantitative and qualitative measurements of host immune responses in serum obtained during the first five days after symptoms onset, in two cohorts of nvH1N1 infected patients. Severe patients required hospitalization (n = 20), due to respiratory insufficiency (10 of them were admitted to the intensive care unit), while mild patients had exclusively flu-like symptoms (n = 15). A group of healthy donors was included as control (n = 15). Differences in levels of mediators between groups were assessed by using the non parametric U-Mann Whitney test. Association between variables was determined by calculating the Spearman correlation coefficient. Viral load was performed in serum by using real-time PCR targeting the neuraminidase gene. RESULTS: Increased levels of innate-immunity mediators (IP-10, MCP-1, MIP-1beta), and the absence of anti-nvH1N1 antibodies, characterized the early response to nvH1N1 infection in both hospitalized and mild patients. High systemic levels of type-II interferon (IFN-gamma) and also of a group of mediators involved in the development of T-helper 17 (IL-8, IL-9, IL-17, IL-6) and T-helper 1 (TNF-alpha, IL-15, IL-12p70) responses were exclusively found in hospitalized patients. IL-15, IL-12p70, IL-6 constituted a hallmark of critical illness in our study. A significant inverse association was found between IL-6, IL-8 and PaO2 in critical patients. CONCLUSIONS: While infection with the nvH1N1 induces a typical innate response in both mild and severe patients, severe disease with respiratory involvement is characterized by early secretion of Th17 and Th1 cytokines usually associated with cell mediated immunity but also commonly linked to the pathogenesis of autoimmune/inflammatory diseases. The exact role of Th1 and Th17 mediators in the evolution of nvH1N1 mild and severe disease merits further investigation as to the detrimental or beneficial role these cytokines play in severe illness.