High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro.

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

Association of hospital contact precaution policies with emergency department admission time.

BACKGROUND: Contact precautions are a widely accepted strategy to reduce in-hospital transmission of meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). However, these practices may have unintended deleterious effects on patients. AIM: To evaluate the effect of a modification in hospital-wide contact precaution practices on emergency department (ED) admission times. METHODS: During the study period, the hospital changed its contact precaution policy from requiring contact precautions for all patients with a history of MRSA or VRE to only those who presented with clinical conditions likely to contaminate the environment with pathogens. An interrupted time series analysis of ED admission times for adults for one year preceding and one year following this change was performed at a two-campus hospital. The main outcome was admission time, defined as time from decision to admit to arrival in an inpatient bed, for patients with MRSA or VRE compared with all other patients. The in-hospital MRSA and VRE acquisition rates were evaluated over the same period and have been published previously. FINDINGS: At one campus, admission time decreased immediately by 161min for MRSA patients (P=0.008) and 135min for VRE patients (P=0.003), and both continued to decrease over the duration of the study. There was no significant change in admission time at the second campus. CONCLUSIONS: Modifying contact precaution requirements for MRSA and VRE may be associated with improved ED admission time without significantly altering in-hospital MRSA and VRE acquisition.

Impact of contact precautions on falls, pressure ulcers and transmission of MRSA and VRE in hospitalized patients.

BACKGROUND: Hospitals use contact precautions to prevent the spread of meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). There is concern that contact precautions may have adverse effects on the safety of isolated patients. In November 2010, the infection control policy at an academic medical centre was modified, and contact precautions were discontinued for patients colonized or infected with MRSA or VRE (MRSA/VRE patients). AIM: To assess the rates of falls and pressure ulcers among MRSA/VRE patients and other adult medical-surgical patients, as well as changes in MRSA and VRE transmission before and after the policy change. METHODS: A single-centre retrospective hospital-wide cohort study was performed from 1st November 2009 to 31st October 2011. FINDINGS: Rates of falls and pressure ulcers were significantly higher among MRSA/VRE patients compared with other adult medical-surgical patients before the policy change (falls: 4.57 vs 2.04 per 1000 patient-days, P < 0.0001; pressure ulcers: 4.87 vs 1.22 per 1000 patient-days, P < 0.0001) and after the policy change (falls: 4.82 vs 2.10 per 1000 patient-days, P < 0.0001; pressure ulcers: 4.17 vs 1.19 per 1000 patient-days, P < 0.0001). No significant differences in the rates of falls and pressure ulcers among MRSA/VRE patients were found after the policy change compared with before the policy change. There was no overall change in MRSA or VRE hospital-acquired transmission. CONCLUSION: MRSA/VRE patients had higher rates of falls and pressure ulcers compared with other adult medical-surgical patients. Rates were not affected by removal of contact precautions, suggesting that other factors contribute to these complications. Further research is required among this population to prevent complications.

Human monoclonal antibody MBL-HCV1 delays HCV viral rebound following liver transplantation: a randomized controlled study.

Rapid allograft infection complicates liver transplantation (LT) in patients with hepatitis C virus (HCV). Pegylated interferon-α and ribavirin therapy after LT has significant toxicity and limited efficacy. The effect of a human monoclonal antibody targeting the HCV E2 glycoprotein (MBL-HCV1) on viral clearance was examined in a randomized, double-blind, placebo-controlled pilot study in patients infected with HCV genotype 1a undergoing LT. Subjects received 11 infusions of 50 mg/kg MBL-HCV1 (n=6) or placebo (n=5) intravenously with three infusions on day of transplant, a single infusion on days 1 through 7 and one infusion on day 14 after LT. MBL-HCV1 was well-tolerated and reduced viral load for a period ranging from 7 to 28 days. Median change in viral load (log10 IU/mL) from baseline was significantly greater (p=0.02) for the antibody-treated group (range -3.07 to -3.34) compared to placebo group (range -0.331 to -1.01) on days 3 through 6 posttransplant. MBL-HCV1 treatment significantly delayed median time to viral rebound compared to placebo treatment (18.7 days vs. 2.4 days, p<0.001). As with other HCV monotherapies, antibody-treated subjects had resistance-associated variants at the time of viral rebound. A combination study of MBL-HCV1 with a direct-acting antiviral is underway.

Impact of toll-like receptor 4 on the severity of acute pancreatitis and pancreatitis-associated lung injury in mice.

BACKGROUND AND AIMS: Acute pancreatitis is an inflammatory disease involving acinar cell injury, and the rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. Toll-like receptor 4 (TLR4) initiates a complex signalling pathway when it interacts with lipopolysaccharide (LPS), which ultimately results in a proinflammatory response. We hypothesised that TLR4 is important in the pathophysiology of acute pancreatitis, independently of LPS. Using two different models of acute pancreatitis, we investigated how genetic deletion of TLR4 or its co-receptor CD14 effects its progression and severity. METHODS: We induced acute pancreatitis by administering either caerulein or L-arginine to wild-type, TLR4(-/-), and CD14(-/-) mice. Control mice received normal saline injections. The severity of acute pancreatitis was determined by measuring serum amylase activity, quantifying myeloperoxidase (MPO) activity in the pancreatic tissue, and histologically assessing acinar cell injury. RESULTS: It was found that administering caerulein and L-arginine to wild-type mice resulted in acute pancreatitis (as assessed by hyperamylasaemia, oedema, increased pancreatic MPO activity, and pancreatic necrosis) and associated lung injury. The same treatment to TLR4(-/-) or CD14(-/-) mice resulted in significantly less severe acute pancreatitis, and reduced lung injury. We found no evidence of either bacteria or LPS in the blood or in pancreatic tissue. CONCLUSIONS: The severity of acute pancreatitis is ameliorated in mice that lack either TLR4 or CD14 receptors. Furthermore, these results indicate that TLR4 plays a significant pro-inflammatory role independently of LPS in the progression of acute pancreatitis.

A transgenic mouse model for studying the clearance of blood-borne pathogens via human complement receptor 1 (CR1).

Complement receptor 1 (CR1) on the surface of human erythrocytes facilitates intravascular clearance of complement-opsonized pathogens. The need for complement activation can be circumvented by directly coupling the organism to CR1 using a bispecific monoclonal antibody heteropolymer (HP). Lack of a functional homologue to CR1 on mouse erythrocytes has made it difficult to study HP-dependent clearance of pathogens in small animals. We have developed a transgenic mouse that expresses human CR1 on erythrocytes. CR1 antigen is of appropriate size and in a clustered distribution as confirmed by immunoblotting and fluorescence microscopy, respectively. HP that immobilized bacteriophage PhiX174 prototype pathogen to erythrocyte CR1 of the transgenic mice increased the rate of clearance of the virus compared with HP that bound bacteriophage, but not CR1. This transgenic mouse model will allow evaluation of different HPs for their in vivo efficacy and potential as human therapeutics.

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.

Epstein-Barr virus-specific T cells for the management of Epstein-Barr virus lymphomas.

Epstein-Barr virus (EBV), a human herpes virus, is associated with a variety of malignancies. In vitro, it is able to transform B cells, which will grow as lymphoblastoid cell lines in the absence of T cells. Patients with a variety of immunodeficiency diseases are subject to the development of B-cell lymphomas that express viral antigens on their cell surface. Development of EBV-associated B-cell lymphomas is seen in solid organ transplant and bone marrow transplant recipients receiving immunosuppressive therapy. Transfer of mature T cells from EBV-immune marrow donors has been demonstrated to be effective in controlling these EBV-associated B-cell tumors. Recently the demonstration that EBV transcripts are found in other lymphomas (including Hodgkin disease cells) has led to the suggestion that transfer of EBV-specific T cells may also be effective in managing these tumors. Current research involves optimizing methods to expand cells that recognize the EBV antigens expressed in the lymphoma cells.

Decreased immediate inflammatory gene induction in activating transcription factor-2 mutant mice.

Transcription factor activating transcription factor (ATF)-2 is activated by inflammatory signals transduced by the JNK and p38 MAP kinase pathways. To better define the role of ATF-2 in inflammation, adult mice expressing small amounts of a mutant ATF-2 protein were challenged with lipopolysaccharide (LPS), anti-CD3 antibody or virus. Within 3 h of challenge by LPS, ATF-2 mutant mice had decreased induction of the adhesion molecules E-selectin, P-selectin and VCAM-1 as well as the cytokines tumor necrosis factor-alpha, IL-1beta and IL-6 compared with control mice. Stimulation of T lymphocytes by anti-CD3 antibody also showed less induction of IL-1 and IL-6 in ATF-2 mutant tissues. ATF-2 mutant thymocytes treated with anti-CD3 antibody in vitro demonstrated reduced induction of c-Jun, JunB, JunD and Fra-2. However, similar to what was observed after p38 kinase inhibition in normal mice, relative ATF-2 deficiency did not prevent the development of a mononuclear cell infiltrate in the week following an inflammatory stimulus. ATF-2 mutant mice proved more susceptible to death than control mice from LPS plus D-galactosamine injection or Coxsackievirus B3 infection and had a higher incidence of mononuclear pulmonary infiltrates after exposure to Herpes simplex virus-1. ATF-2 is essential for maximal immediate induction of adhesion molecules and cytokine genes, but at later time points may even protect against overactive immune responses.

Bacterial pathogens induce abscess formation by CD4(+) T-cell activation via the CD28-B7-2 costimulatory pathway.

Abscesses are a classic host response to infection by many pathogenic bacteria. The immunopathogenesis of this tissue response to infection has not been fully elucidated. Previous studies have suggested that T cells are involved in the pathologic process, but the role of these cells remains unclear. To delineate the mechanism by which T cells mediate abscess formation associated with intra-abdominal sepsis, the role of T-cell activation and the contribution of antigen-presenting cells via CD28-B7 costimulation were investigated. T cells activated in vitro by zwitterionic bacterial polysaccharides (Zps) known to induce abscess formation required CD28-B7 costimulation and, when adoptively transferred to the peritoneal cavity of naïve rats, promoted abscess formation. Blockade of T-cell activation via the CD28-B7 pathway in animals with CTLA4Ig prevented abscess formation following challenge with different bacterial pathogens, including Staphylococcus aureus, Bacteroides fragilis, and a combination of Enterococcus faecium and Bacteroides distasonis. In contrast, these animals had an increased abscess rate following in vivo T-cell activation via CD28 signaling. Abscess formation in vivo and T-cell activation in vitro required costimulation by B7-2 but not B7-1. These results demonstrate that abscess formation by pathogenic bacteria is under the control of a common effector mechanism that requires T-cell activation via the CD28-B7-2 pathway.

Detection of Gov system antibodies by MAIPA reveals an immunogenicity similar to the HPA-5 alloantigens.

The glycosylphosphatidylinositol-linked platelet protein CD109 carries the biallelic alloantigen system Gov. There is limited information on the incidence of Gov alloantibodies in neonatal alloimmune thrombocytopenia (NAITP), post-transfusion purpura (PTP) and platelet refractoriness. We adapted the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay to the detection of Gov antibodies and determined their incidence in 605 archived samples (112 with HPA antibodies) referred for the aforementioned conditions. Here, we show that CD109 expression was reduced upon platelet storage in saline or by cryopreservation, but was stable when stored as whole blood or therapeutic platelet concentrate. Fourteen of the 605 samples contained Gov alloantibodies (anti-Gova, n = 10; anti-Govb, n = 4), with the majority in platelet refractoriness (n = 9) and, of the remaining five, four in NAITP and one in PTP. In seven cases, no other HPA antibodies were detected, three being NAITP cases. The incidence of Gov antibodies was significantly lower than HPA-1 system antibodies (n = 87), but equalled the number of HPA-5 system antibodies (n = 14) and outnumbered HPA-2 and -3 system antibodies (10 altogether).

Maturation and lineage-specific expression of the coxsackie and adenovirus receptor in hematopoietic cells.

Adenovirus vectors have been used to transfer genes into both hematopoietic progenitor cells and tumor cells, including carcinoma cells that have metastasized to bone marrow (BM). However, the relative susceptibility of different subsets of hematopoietic cells is unknown. In permissive cells adenoviral-mediated gene transfer is mediated by the coxsackievirus and adenovirus receptor (CAR) protein and alpha(v) integrins expressed on the cell surface of the target cells. This prompted us to investigate the expression of CAR on subpopulations of hematopoietic cells, determine whether this protein played a role in adenovirus-mediated gene transfer of hematopoietic cells and whether we could modulate CAR to enhance gene transfer efficiency. In this report we show that CAR is expressed on approximately 40% of all human BM cells, including erythroid and myeloid cells, but not lymphoid cells. Of the CD34(+) cells, 10%-15% expressed CAR, but this did not include most colony-forming progenitor cells, nor the most primitive CD38(-) subpopulation. The presence of CAR correlated well with gene transfer efficiency, but we were unable to induce CAR expression on immature, noncommitted progenitor cells. In conclusion, our results show that primitive hematopoietic progenitor cells lack CAR expression, but that expression is acquired during erythroid and myeloid differentiation.

The coxsackie-adenovirus receptor (CAR) is used by reference strains and clinical isolates representing all six serotypes of coxsackievirus group B and by swine vesicular disease virus.

Group B coxsackieviruses are etiologically linked to many human diseases, and cell surface receptors are postulated to play an important role in mediating their pathogenesis. The coxsackievirus adenovirus receptor (CAR) has been shown to function as a receptor for selected strains of coxsackievirus group B (CVB) serotypes 3, 4, and 5 and is postulated to serve as a receptor for all six serotypes. In this study, we demonstrate that CAR can serve as a receptor for laboratory reference strains and clinical isolates of all six CVB serotypes. Infection of CHO cells expressing human CAR results in a 1000-fold increase in CVB progeny virus titer compared to mock transfected cells. CAR was shown to be a functional receptor for swine vesicular disease virus (SVDV), as CHO-CAR cells but not CHO mock transfected controls were susceptible to SVDV infection, produced progeny SVDV, and developed cytopathic effects. Moreover, SVDV infection could be specifically blocked by monoclonal antibody to CAR (RmcB). SVDV infection of HeLa cells was also inhibited by an anti-CD55 MAb, suggesting that this virus, like some CVB, may interact with CD55 (decay accelerating factor) in addition to CAR. Finally, pretreatment of CVB or SVDV with soluble CAR effectively blocks virus infection of HeLa cell monolayers.

T cells activated by zwitterionic molecules prevent abscesses induced by pathogenic bacteria.

Immunologic paradigms classify bacterial polysaccharides as T cell-independent antigens. However, these models fail to explain how zwitterionic polysaccharides (Zps) confer protection against intraabdominal abscess formation in a T cell-dependent manner. Here, we demonstrate that Zps elicit a potent CD4+ T cell response in vitro that requires available major histocompatibility complex class II molecules on antigen-presenting cells. Specific chemical modifications to Zps show that: 1) the activity is specific for carbohydrate structure, and 2) the proliferative response depends upon free amino and carboxyl groups on the repeating units of these polysaccharides. Peptides synthesized to mimic the zwitterionic charge motif associated with Zps also exhibited these biologic properties. Lysine-aspartic acid (KD) peptides with more than 15 repeating units stimulated CD4+ T cells in vitro and conferred protection against abscesses induced by bacteria such as Bacteroides fragilis and Staphylococcus aureus. Evidence for the biologic importance of T cell activation by these zwitterionic polymers was provided when human CD4+ T cells stimulated with these molecules in vitro and adoptively transferred to rats in vivo conferred protection against intraabdominal abscesses induced by viable bacterial challenge. These studies demonstrate that bacterial polysaccharides with a distinct charge motif activate T cells and that this activity confers immunity to a distinct pathologic response to bacterial infection.

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide.

Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.

Effect of molecular size on the ability of zwitterionic polysaccharides to stimulate cellular immunity.

The large-molecular-sized zwitterionic capsular polysaccharide of the anaerobe Bacteroides fragilis NCTC 9343, designated polysaccharide (PS) A, stimulates T cell proliferation in vitro and induces T cell-dependent protection against abscess formation in vivo. In the present study, we utilized a modification of a recently developed ozonolytic method for depolymerizing polysaccharides to examine the influence of the molecular size of PS A on cell-mediated immunity. Ozonolysis successfully depolymerized PS A into structurally intact fragments. PS A with average molecular sizes of 129.0 (native), 77.8, 46.9, and 17.1 kDa stimulated CD4+-cell proliferation in vitro to the same degree, whereas the 5.0-kDa fragment was much less stimulatory than the control 129.0-kDa PS A. Rats treated with 129.0-kDa, 46.9-kDa, and 17.1-kDa PS A molecules, but not those treated with the 5.0-kDa molecule, were protected against intraabdominal abscesses induced by challenge with viable B. fragilis. These results demonstrate that a zwitterionic polysaccharide as small as 22 repeating units (88 monosaccharides) elicits a T cell-dependent immune response. These findings clearly distinguish zwitterionic T cell-dependent polysaccharides from T cell-independent polysaccharides and give evidence of the existence of a novel mechanism for a polysaccharide-induced immune response.

Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells.

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.

Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.

The innate immune system contributes to the earliest phase of the host defense against foreign organisms and has both soluble and cellular pattern recognition receptors for microbial products. Two important members of this receptor group, CD14 and the Toll-like receptor (TLR) pattern recognition receptors, are essential for the innate immune response to components of Gram-negative and Gram-positive bacteria, mycobacteria, spirochetes and yeast. We now find that these receptors function in an antiviral response as well. The innate immune response to the fusion protein of an important respiratory pathogen of humans, respiratory syncytial virus (RSV), was mediated by TLR4 and CD14. RSV persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Thus, a common receptor activation pathway can initiate innate immune responses to both bacterial and viral pathogens.

Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.

Toll-like receptors (TLRs) 2 and 4 are signal transducers for lipopolysaccharide, the major proinflammatory constituent in the outer membrane of Gram-negative bacteria. We observed that membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans activated cells heterologously expressing TLR2 but not those expressing TLR1 or TLR4. These TLR2-expressing cells were also stimulated by living motile B. burgdorferi, suggesting that TLR2 recognition of lipoproteins is relevant to natural Borrelia infection. Importantly, a TLR2 antibody inhibited bacterial lipoprotein/lipopeptide-induced tumor necrosis factor release from human peripheral blood mononuclear cells, and TLR2-null Chinese hamster macrophages were insensitive to lipoprotein/lipopeptide challenge. The data suggest a role for the native protein in cellular activation by these ligands. In addition, TLR2-dependent responses were seen using whole Mycobacterium avium and Staphylococcus aureus, demonstrating that this receptor can function as a signal transducer for a wide spectrum of bacterial products. We conclude that diverse pathogens activate cells through TLR2 and propose that this molecule is a central pattern recognition receptor in host immune responses to microbial invasion.