Recruitment of monocytes primed to express heme oxygenase-1 ameliorates pathological lung inflammation in cystic fibrosis.

Overwhelming neutrophilic inflammation is a leading cause of lung damage in many pulmonary diseases, including cystic fibrosis (CF). The heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway mediates the resolution of inflammation and is defective in CF-affected macrophages (MΦs). Here, we provide evidence that systemic administration of PP-007, a CO releasing/O2 transfer agent, induces the expression of HO-1 in a myeloid differentiation factor 88 (MyD88) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)-dependent manner. It also rescues the reduced HO-1 levels in CF-affected cells induced in response to lipopolysaccharides (LPS) or Pseudomonas aeruginosa (PA). Treatment of CF and muco-obstructive lung disease mouse models with a single clinically relevant dose of PP-007 leads to effective resolution of lung neutrophilia and to decreased levels of proinflammatory cytokines in response to LPS. Using HO-1 conditional knockout mice, we show that the beneficial effect of PP-007 is due to the priming of circulating monocytes trafficking to the lungs in response to infection to express high levels of HO-1. Finally, we show that PP-007 does not compromise the clearance of PA in the setting of chronic airway infection. Overall, we reveal the mechanism of action of PP-007 responsible for the immunomodulatory function observed in clinical trials for a wide range of diseases and demonstrate the potential use of PP-007 in controlling neutrophilic pulmonary inflammation by promoting the expression of HO-1 in monocytes/macrophages.

Microvascular and Systemic Impact of Resuscitation with PEGylated Carboxyhemoglobin-Based Oxygen Carrier or Hetastarch in a Rat Model of Transient Hemorrhagic Shock.

BACKGROUND: Hemorrhage is the leading cause of preventable, traumatic death. Currently, prehospital resuscitation fluids provide preload but not oxygen-carrying capacity-a critical blood function that mitigates microvascular ischemia and tissue hypoxia during hemorrhagic shock. Solutions containing polymerized hemoglobin have been associated with vasoactive and hypertensive events. A novel hemoglobin-based oxygen carrier, modified with PEGylation and CO moieties (PEG-COHb), may overcome these limitations. OBJECTIVES: To evaluate the systemic and microcirculatory effects of PEG-COHb as compared with the 6% hetastarch in a rat model of hemorrhagic shock. METHODS: Male Sprague Dawley rats (N = 20) were subjected to severe, controlled, hemorrhagic shock. Animals were randomized to 20% estimated blood-volume resuscitation with either 6% hetastarch or PEG-COHb. Continuous, invasive, cardiovascular measurements, and arterial blood gases were measured. Microcirculatory measurements of interstitial oxygenation (PISFO2) and vasoactivity helped model oxygen delivery in the spinotrapezius muscle using intravital and phosphorescence quenching microscopy. RESULTS: Hemorrhage reduced mean arterial pressure (MAP), arteriolar diameter, and PISFO2, and increased lactate 10-fold in both groups. Resuscitation with both PEG-COHb and hetastarch improved cardiovascular parameters. However, PEG-COHb treatment resulted in higher MAP (P < 0.001), improved PISFO2 (14 [PEG-COHb] vs. 5 [hetastarch] mmHg; P < 0.0001), lower lactate post-resuscitation (P < 0.01), and extended survival from 90 to 142 min (P < 0.001) as compared with the hetastarch group. CONCLUSIONS: PEG-COHb improved MAP PISFO2, lactate, and survival time as compared with 6% hetastarch resuscitation. Importantly, hypertension and vasoactivity were not detected in response to PEG-COHb resuscitation supporting further investigation of this resuscitation strategy.

Effect of Polyethylene-glycolated Carboxyhemoglobin on Renal Microcirculation in a Rat Model of Hemorrhagic Shock.

BACKGROUND: Primary resuscitation fluid to treat hemorrhagic shock remains controversial. Use of hydroxyethyl starches raised concerns of acute kidney injury. Polyethylene-glycolated carboxyhemoglobin, which has carbon monoxide-releasing molecules and oxygen-carrying properties, was hypothesized to sustain cortical renal microcirculatory PO2 after hemorrhagic shock and reduce kidney injury. METHODS: Anesthetized and ventilated rats (n = 42) were subjected to pressure-controlled hemorrhagic shock for 1 h. Renal cortical PO2 was measured in exposed kidneys using a phosphorescence quenching method. Rats were randomly assigned to six groups: polyethylene-glycolated carboxyhemoglobin 320 mg · kg, 6% hydroxyethyl starch (130/0.4) in Ringer's acetate, blood retransfusion, diluted blood retransfusion (~4 g · dl), nonresuscitated animals, and time control. Nitric oxide and heme oxygenase 1 levels were determined in plasma. Kidney immunohistochemistry (histologic scores of neutrophil gelatinase-associated lipocalin and tumor necrosis factor-α) and tubular histologic damages analyses were performed. RESULTS: Blood and diluted blood restored renal PO2 to 51 ± 5 mmHg (mean difference, -18; 95% CI, -26 to -11; P < 0.0001) and 47 ± 5 mmHg (mean difference, -23; 95% CI, -31 to -15; P < 0.0001), respectively, compared with 29 ± 8 mmHg for hydroxyethyl starch. No differences between polyethylene-glycolated carboxyhemoglobin and hydroxyethyl starch were observed (33 ± 7 mmHg vs. 29 ± 8 mmHg; mean difference, -5; 95% CI, -12 to 3; P = 0.387), but significantly less volume was administered (4.5 [3.3-6.2] vs. 8.5[7.7-11.4] ml; mean rank difference, 11.98; P = 0.387). Blood and diluted blood increased the plasma bioavailability of nitric oxide compared with hydroxyethyl starch (mean rank difference, -20.97; P = 0.004; and -17.13; P = 0.029, respectively). No changes in heme oxygenase 1 levels were observed. Polyethylene-glycolated carboxyhemoglobin limited tubular histologic damages compared with hydroxyethyl starch (mean rank difference, 60.12; P = 0.0012) with reduced neutrophil gelatinase-associated lipocalin (mean rank difference, 84.43; P < 0.0001) and tumor necrosis factor-α (mean rank difference, 49.67; P = 0.026) histologic scores. CONCLUSIONS: Polyethylene-glycolated carboxyhemoglobin resuscitation did not improve renal PO2 but limited tubular histologic damages and neutrophil gelatinase-associated lipocalin upregulation after hemorrhage compared with hydroxyethyl starch, whereas a lower volume was required to sustain macrocirculation.

Microvascular and systemic responses to novel PEGylated carboxyhaemoglobin-based oxygen carrier in a rat model of vaso-occlusive crisis.

Hypoxia drives sickle cell disease (SCD) by inducing sickle cell haemoglobin to polymerize and deform red blood cells (RBC) into the sickle shape. A novel carboxyhaemoglobin-based oxygen carrier (PEG-COHb; PP-007) promotes unsickling in vitro by relieving RBC hypoxia. An in vivo rat model of vaso-occlusive crisis (VOC) capable of accommodating a suite of physiological and microcirculatory measurements was used to compare treatment with PEG-COHb to a non-oxygen carrying control solution (lactated ringer's [LRS]). Male Sprague-Dawley rats were anesthetized and surgically prepared to monitor microvascular interstitial oxygenation (PISFO2), cardiovascular parameters and blood chemistry. Human homozygous SCD RBCs were isolated and exchange transfused into the rats until the distal microcirculation of the exteriorized spinotrapezius muscle was hypoxic and RBC aggregates were visualized. VOC was left untreated (Sham) or treated 15 min later with PEG-COHb or LRS and observed for up to 4 h. Treatment with PEG-COHb showed better improvement of PISFO2, end-point lactate, mean arterial pressure and survival duration compared to Sham and LRS. Restoring PISFO2 was associated with relieving the RBC aggregates driving VOC, which then affected other study metrics. Compared to LRS, PEG-COHb's oxygen-carrying properties were key to improved outcomes.

Effects of Sanguinate on Systemic and Microcirculatory Variables in a Model of Prolonged Hemorrhagic Shock.

BACKGROUND: Hemorrhage and its complications are the leading cause of preventable death from trauma in young adults, especially in remote locations. To address this, deliverable, shelf-stable resuscitants that provide therapeutic benefits throughout the time course of hemorrhagic shock and the progressive ischemic injury it produces are needed. SANGUINATE is a novel bovine PEGylated carboxyhemoglobin-based oxygen carrier, which has desirable oxygen-carrying and oncotic properties as well as a CO moiety to maintain microvascular perfusion. OBJECTIVES: To compare the crystalloid (Lactated Ringer's Solution; LRS), and the colloid (Hextend) standards of care with SANGUINATE in a post "golden hour" resuscitation model. METHODS: Rats underwent a controlled, stepwise blood withdrawal (45% by volume), were maintained in untreated hemorrhagic shock state for >60 min, resuscitated with a 20% bolus of one of the three test solutions, and observed till demise. Parameters of tissue oxygenation (PISFO2), arteriolar diameters, and mean arterial pressure (MAP) were collected. RESULTS: SANGUINATE-treated animals survived significantly longer than those treated with Hextend and LRS. SANGUINATE also significantly increased tissue PISFO2 2 h after resuscitation, whereas LRS and Hextend did not. SANGUINATE also produced a significantly higher MAP, which was hypotensive compared to baseline, that endured until demise. CONCLUSIONS: Resuscitation with SANGUINATE after prolonged hemorrhagic shock improves survival, MAP, and PISFO2 compared with standard of care plasma expanders. Since the pathologies of hemorrhagic shock and the associated systemic ischemia are progressive, preclinical studies of this nature are essential to determine efficacy of new resuscitants across the range of possible times to treatment.

A Phase I study to determine safety, pharmacokinetics, and pharmacodynamics of ANF-RHO™, a novel PEGylated granulocyte colony-stimulating factor, in healthy volunteers.

Patients receiving pegfilgrastim (Neulasta®) for the treatment of neutropenia can experience bone pain following the injections required to achieve effective neutrophil levels. The safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles of ANF-RHO™, a novel pegylated granulocyte colony stimulating factor, were assessed in a randomized, controlled, double-blind Phase 1 clinical study in healthy volunteers. Subjects received a single subcutaneous dose of ANF-RHO over a range of 6 doses (5-50 µg/kg), placebo (saline), or the recommended clinical dose of pegfilgrastim administered at the labeled fixed 6 mg dosage (equivalent to 80-100 µg/kg). The primary outcome measure was safety and tolerability. Secondary outcomes included PK and PD effects on absolute neutrophil count (ANC) and number of CD34+ progenitor cells. Severity of bone pain was also assessed. In healthy volunteers, ANF-RHO was administered at ascending doses up to 50 µg/kg without significant adverse effects; appeared to be better (5 to 30 µg/kg) or equally well (50 µg/kg) tolerated, and had lower mean bone pain scores as compared to pegfilgrastim. ANF-RHO achieved CD34+ and ANC numbers at significantly lower doses, and had a significantly longer circulating half-life than pegfilgrastim. These results suggest that ANF-RHO can be provided less frequently, at a lower dose, and with fewer side effects. ANF-RHO had unique, prolonged PK/PD attributes as compared to marketed pegfilgrastim, suggesting that it may provide an improved clinical benefit in further clinical studies in patients with chemotherapy-induced or chronic idiopathic neutropenia.

Pharmacologically increasing collateral perfusion during acute stroke using a carboxyhemoglobin gas transfer agent (Sanguinate™) in spontaneously hypertensive rats.

Similar to patients with chronic hypertension, spontaneously hypertensive rats (SHR) develop fast core progression during middle cerebral artery occlusion (MCAO) resulting in large final infarct volumes. We investigated the effect of Sanguinate™ (SG), a PEGylated carboxyhemoglobin (COHb) gas transfer agent, on changes in collateral and reperfusion cerebral blood flow and brain injury in SHR during 2 h of MCAO. SG (8 mL/kg) or vehicle ( n = 6-8/group) was infused i.v. after 30 or 90 min of ischemia with 2 h reperfusion. Multi-site laser Doppler probes simultaneously measured changes in core MCA and collateral flow during ischemia and reperfusion using a validated method. Brain injury was measured using TTC. Animals were anesthetized with choral hydrate. Collateral flow changed little in vehicle-treated SHR during ischemia (-8 ± 9% vs. prior to infusion) whereas flow increased in SG-treated animals (29 ± 10%; p < 0.05). In addition, SG improved reperfusion regardless of time of treatment; however, brain injury was smaller only with early treatment in SHR vs. vehicle (28.8 ± 3.2% vs. 18.8 ± 2.3%; p < 0.05). Limited collateral flow in SHR during MCAO is consistent with small penumbra and large infarction. The ability to increase collateral flow in SHR with SG suggests that this compound may be useful as an adjunct to endovascular therapy and extend the time window for treatment.

Respiratory syncytial virus infection induces a subset of types I and III interferons in human dendritic cells.

Whether respiratory syncytial virus (RSV) induces severe infantile pulmonary disease may depend on viral strain and expression of types I and III interferons (IFNs). These IFNs impact disease severity by inducing expression of many anti-viral IFN-stimulated genes (ISGs). To investigate the impact of RSV strain on IFN and ISG expression, we stimulated human monocyte-derived DCs (MDDCs) with either RSV A2 or Line 19 and measured expression of types I and III IFNs and ISGs. At 24h, A2 elicited higher ISG expression than Line 19. Both strains induced MDDCs to express genes for IFN-ß, IFN-α1, IFN-α8, and IFN-λ1-3, but only A2 induced IFN-α2, -α14 and -α21. We then show that IFN-α8 and IFN-α14 most potently induced MDDCs and bronchial epithelial cells (BECs) to express ISGs. Our findings demonstrate that RSV strain may impact patterns of types I and III IFN expression and the magnitude of the ISG response by DCs and BECs.

Chemotherapeutics and radiation stimulate MHC class I expression through elevated interferon-beta signaling in breast cancer cells.

Low doses of anticancer drugs have been shown to enhance antitumor immune response and increase the efficacy of immunotherapy. The molecular basis for such effects remains elusive, although selective depletion of T regulatory cells has been demonstrated. In the current studies, we demonstrate that topotecan (TPT), a topoisomerase I-targeting drug with a well-defined mechanism of action, stimulates major histocompatibility complex class I (MHC I) expression in breast cancer cells through elevated expression/secretion of interferon-ß (IFN-ß) and activation of type I IFN signaling. First, we show that TPT treatment elevates the expression of both total and cell-surface MHC I in breast cancer cells. Second, conditioned media from TPT-treated breast cancer ZR-75-1 cells induce elevated expression of cell-surface MHC I in drug-naïve recipient cells, suggesting the involvement of cytokines and/or other secreted molecules. Consistently, TPT-treated cells exhibit elevated expression of multiple cytokines such as IFN-ß, TNF-α, IL-6 and IL-8. Third, either knocking down the type I interferon receptor subunit 1 (IFNAR1) or addition of neutralizing antibody against IFN-ß results in reduced MHC I expression in TPT-treated cells. Together, these results suggest that TPT induces increased IFN-ß autocrine/paracrine signaling through type I IFN receptor, resulting in the elevated MHC I expression in tumor cells. Studies have also demonstrated that other chemotherapeutic agents (e.g. etoposide, cisplatin, paclitaxel and vinblastine) similarly induce increased IFN-ß secretion and elevated MHC I expression. In addition, conditioned media from γ-irradiated donor cells are shown to induce IFN-ß-dependent MHC I expression in unirradiated recipient cells. In the aggregate, our results suggest that many cancer therapeutics induce elevated tumor antigen presentation through MHC I, which could represent a common mechanism for enhanced antitumor immune response through T cell cytotoxicity during metronomic chemotherapy, as well as increased efficacy of combined chemo- (or radio-)/immuno-therapy.

Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent.

Recent genome-wide association studies suggest distinct roles for 12 human interferon-alpha (IFN-α) and 3 IFN-λ subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides. We found that in response to poly I:C and LPS, monocytes, MDM and MDDC express a subtype pattern restricted primarily to IFN-ß and IFN-λ1. In addition, while CpG elicited expression of all type I IFN subtypes by pDC, imiquimod did not. Furthermore, MDM and mDC highly express IFN-λ, and the subtypes of IFN-λ are expressed hierarchically in the order IFN-λ1 followed by IFN-λ2, and then IFN-λ3. These data support a model of coordinated cell- and ligand-specific expression of types I and III IFN. Defining IFN subtype expression profiles in a variety of contexts may elucidate specific roles for IFN subtypes as protective, therapeutic or pathogenic mediators.

Interferon ß is associated with type 1 interferon-inducible gene expression in dermatomyositis.

OBJECTIVES: To determine whether type 1 interferon (IFN) proteins in blood are associated with downstream type 1 IFN-inducible gene expression in blood from patients with myositis. METHODS: IFNα, IFNß and IFNω concentrations were measured by ELISA in 129 blood samples (from 93 patients with dermatomyositis (DM), inclusion body myositis, polymyositis and other muscle diseases and from 36 healthy volunteers). Their concentrations were correlated with their ability to stimulate type 1 IFN-inducible gene transcription in a functional assay for 123 of these samples and the type 1 IFN-inducible blood gene expression from 70 of the same samples. RESULTS: Blood IFNß concentration was uniquely associated with DM (p=0.0004), detectable in 64% of samples from patients with untreated or minimally treated DM and 35% of all DM samples compared with 6% of other inflammatory myopathy and 6% of healthy volunteer samples. Blood IFNß, but not IFNα or IFNω, correlated with high blood type 1 IFN-inducible gene expression (p=0.01). Healthy volunteer samples with a high ELISA signal for IFNα and IFNω lacked functional bioassay activity and such a signal was confirmed as artefactual. CONCLUSION: Elevated blood IFNß protein concentration is associated with DM. Systemic and local production of IFNß might contribute to, but may not fully explain, the marked overproduction of type 1 IFN-inducible transcripts and proteins seen in DM muscle and blood.

Human plasmacytoid dendritic cell accumulation amplifies their type 1 interferon production.

To determine the potential consequences of plasmacytoid dendritic cell (pDC) accumulation in tissue sites observed in several autoimmune diseases, we measured type 1 interferon production from circulating human pDCs as a function of pDC concentration. The effects of interferon-alpha and blockade of the type 1 interferon receptor (IFNAR) on human pDC type 1 interferon and interferon-inducible transcription and protein production were measured. Human pDCs became far more efficient producers of interferon-alpha at concentrations beyond those normally present in blood, through an IFNAR-dependent mechanism. Extracellular interferon-alpha increased pDC production of type 1 interferons. The accumulation of pDCs in diseased tissue sites allows marked non-linear amplification of type 1 interferon production locally. The role of the IFNAR-dependent mechanism of interferon production by human pDCs is greater than previously suggested. IFNAR blockade has potential for diminishing type 1 interferon production by all human cells.

Antiviral activity of transiently expressed IFN-kappa is cell-associated.

Most type I interferons (IFNs) are expressed by the majority of cell types in response to viral infection. In contrast, IFN-kappa has been reported to have a cellular distribution limited to keratinocytes and certain lymphoid cell populations. Recombinant expressed IFN-kappa has been shown previously to possess weak antiviral activity when directly compared with IFN-beta. In order to expand on the antiviral potential of IFN-kappa, we transiently transfected human cell lines to circumvent the need to purify recombinant proteins and to avoid the possible loss of biologic activity by the purification process. We evaluated the transcriptional signaling and antiviral activity of IFN-kappa in parallel with IFN-alpha2b with mammalian expression vectors to express each protein transiently. Both IFN-kappa and IFN-alpha2b exhibited comparable transcriptional and antiviral activities. However, in contrast to IFN-alpha2b transcriptional signaling and antiviral activity, IFN-kappa activity was not detectable in conditioned cell culture medium. Subsequent experiments revealed there was a direct relationship between IFN-kappa-expressing cells and antiviral activity. These results were confirmed in immunocytochemical studies. Furthermore, IFN-kappa exhibited cell-associated antiviral activity against a hepatitis C virus (HCV) replicon cell line. This novel IFN signaling strategy may represent an important distinct and divergent mechanism for limiting viral infections.

Targeting hepatitis C virus translation: stopping HCV where it starts.

Hepatitis C virus (HCV) infection is an emerging global health concern. Combination therapy with pegylated interferon-alpha (INF alpha) and ribavirin results in approximately 60% sustained recovery. Of the non-responders, the majority are infected with genotype 1. As a consequence, improved therapeutics are necessary to enhance response rates, especially in genotype-1-infected individuals. HCV translation is mediated by an internal ribosome entry site (IRES) located within the 5' non-translated region. Recent studies have revealed that the HCV IRES recruits the cellular translation machinery in a distinct manner compared with cellular mRNA strategies. Therefore, screening assays can be developed to identify specific inhibitors of HCV translation as a possible treatment for HCV.

Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation.

The hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA drives internal initiation of viral protein synthesis during host cell infection. In the tertiary structure of the IRES RNA, two helical junctions create recognition sites for direct binding of the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). The 2.8 A resolution structure of the IIIabc four-way junction, which is critical for binding eIF3, reveals how junction nucleotides interact with an adjacent helix to position regions directly involved in eIF3 recognition. Two of the emergent helices stack to form a nearly continuous A-form duplex, while stacking of the other two helices is interrupted by the insertion of junction residues into the helix minor groove. This distorted stack probably serves as an important recognition surface for the translational machinery.