Mesenchymal Stromal (Stem) Cell Therapy Fails to Improve Outcomes in Experimental Severe Influenza

Rationale

Severe influenza remains a major public health threat and is responsible for thousands of deaths annually. Increasing antiviral resistance and limited effectiveness of current therapies highlight the need for new approaches to influenza treatment. Extensive pre-clinical data have shown that mesenchymal stromal (stem) cell (MSC) therapy can induce anti-inflammatory effects and enhance repair of the injured lung. We hypothesized that MSC therapy would improve survival, dampen lung inflammation and decrease acute lung injury (ALI) in a murine model of severe influenza.

Methods

C57Bl/6 mice were infected with influenza A/PuertoRico/8/34 (mouse-adapted H1N1) or influenza A/Mexico/4108/2009 (swine-origin pandemic H1N1) and administered human or mouse MSCs via the tail vein, either pre- or post- infection. MSC efficacy was evaluated as both an independent and adjunctive treatment strategy in combination with the antiviral agent, oseltamivir. Weight loss and survival were monitored. Inflammatory cells, cytokine/chemokines (IFN-γ, CXCL10, CCL2 and CCL5) and markers of ALI (total protein and IgM), were measured in bronchoalveolar lavage fluid and lung parenchyma.

Results

Administration of murine MSCs or human MSCs in a prophylactic or therapeutic regimen failed to improve survival, decrease pulmonary inflammation/inflammatory cell counts or prevent ALI in influenza virus-infected mice. MSCs administered in combination with oseltamivir also failed to improve outcomes.

Conclusions

Despite similarities in the clinical presentation and pathobiology of ALI and severe influenza, our findings suggest that MSC therapy may not be effective for prevention and/or treatment of acute severe influenza.     

The optimization of aminooxadiazoles as orally active inhibitors of Cdc7

A series of aminooxadiazoles was optimized for inhibition of Cdc7. Early lead isoquinoline 1 suffered from modest cell potency (cellular IC₅₀ = 0.71 μM measuring pMCM2), low selectivity against structurally related kinases, and high IV clearance in rats (CL = 18 L/h/kg). Extensive optimization resulted in azaindole 26 (Cdc7 IC₅₀ = 1.1 nM, pMCM2 IC₅₀ = 32 nM) that demonstrated robust lowering of pMCM2 in a mouse pharmacodynamic (PD) model when dosed orally. Modifications to improve the pharmacokinetic profile of this series were guided by trapping experiments with glutathione in rat hepatocytes.     

The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed –1 ribosomal frameshifting

Several important viruses including the human immunodeficiency virus type 1 (HIV-1) and the SARS-associated Coronavirus (SARS-CoV) employ programmed −1 ribosomal frameshifting (PRF) for their protein expression. Here, a kinetic framework is developed to describe −1 PRF. The model reveals three kinetic pathways to −1 PRF that yield two possible frameshift products: those incorporating zero frame encoded A-site tRNAs in the recoding site, and products incorporating −1 frame encoded A-site tRNAs. Using known kinetic rate constants, the individual contributions of different steps of the translation elongation cycle to −1 PRF and the ratio between two types of frameshift products were evaluated. A dual fluorescence reporter was employed in Escherichia coli to empirically test the model. Additionally, the study applied a novel mass spectrometry approach to quantify the ratios of the two frameshift products. A more detailed understanding of the mechanisms underlying −1 PRF may provide insight into developing antiviral therapeutics.     

Synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice

The severe acute respiratory syndrome (SARS) epidemic was characterized by high mortality rates in the elderly. The molecular mechanisms that govern enhanced susceptibility of elderly populations are not known, and robust animal models are needed that recapitulate the increased pathogenic phenotype noted with increasing age. Using synthetic biology and reverse genetics, we describe the construction of a panel of isogenic SARS coronavirus (SARS-CoV) strains bearing variant spike glycoproteins that are representative of zoonotic strains found in palm civets and raccoon dogs, as well as isolates spanning the early, middle, and late phases of the SARS-CoV epidemic. The recombinant viruses replicated efficiently in cell culture and demonstrated variable sensitivities to neutralization with antibodies. The human but not the zoonotic variants replicated efficiently in human airway epithelial cultures, supporting earlier hypotheses that zoonotic isolates are less pathogenic in humans but can evolve into highly pathogenic strains. All viruses replicated efficiently, but none produced clinical disease or death in young animals. In contrast, severe clinical disease, diffuse alveolar damage, hyaline membrane formation, alveolitis, and death were noted in 12-month-old mice inoculated with the palm civet HC/SZ/61/03 strain or early-human-phase GZ02 variants but not with related middle- and late-phase epidemic or raccoon dog strains. This panel of SARS-CoV recombinants bearing zoonotic and human epidemic spike glycoproteins will provide heterologous challenge models for testing vaccine efficacy against zoonotic reintroductions as well as provide the appropriate model system for elucidating the complex virus-host interactions that contribute to more-severe and fatal SARS-CoV disease and acute respiratory distress in the elderly.     

Genomics and proteomics in process development: opportunities and challenges

Global gene expression profiling by genomic and proteomic analyses has changed the face of drug discovery and biological research in the past few years. The benefit of these technologies in the area of process development for recombinant protein production has been increasingly realized. This review discusses the application of genome-wide expression profiling tools in the design and optimization of bioprocesses, with the emphasis on the effect on process development of mammalian cell culture. Despite the lack of genome sequence information for most of the relevant mammalian cell lines used, these technologies can be applied during various process development steps. Although there are only a few examples in the literature that present a major improvement in productivity based on genomics and proteomics, further advances in analytical tools and genome sequencing technologies will greatly increase our knowledge at the molecular level and will drive the design of future bioprocesses.     

Application of atmospheric pressure chemical ionisation mass spectrometry in the identification and differentiation of Panax species

An HPLC-MS method using an atmospheric pressure chemical ionisation (APCI) source has been developed to assist in the differentiation of three ginseng species: Panax quinquefolium (American ginseng), P. ginseng (Chinese ginseng) and P. notoginseng (sanqi) species. The differentiation method relies on the identification of ginsenosides Rf and F11 and notoginsenoside R1. R1 is observed in both P. notoginseng and Chinese ginseng, whilst F1, is found exclusively in the American species. The presence of these compounds permits the definitive identification of the species to be made. The APCI ionisation source has been employed to tackle the matrix interference in analysing Chinese medicinal materials and to minimise the associated matrix effects that are commonly encountered with other ionisation modes. Moreover, the method allows direct interface to conventional HPLC systems. More importantly, chemical reference standards of ginsenosides are not required in this method. This technique provides an alternative approach to analysing high molecular weight polar compounds that typically encountered in complex matrices of Chinese medicinal materials.     

The C-terminal aqueous-exposed domain of the 45 kDa subunit of the particulate methane monooxygenase in Methylococcus capsulatus (Bath) is a Cu(I) sponge

The crystal structure of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) has been reported recently [Lieberman, R. L., and Rosenzweig, A. C. (2005) Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane, Nature 434, 177-182]. Subsequent work has shown that the preparation on which the X-ray analysis is based might be missing many of the important metal cofactors, including the putative trinuclear copper cluster at the active site as well as ca. 10 copper ions (E-clusters) that have been proposed to serve as a buffer of reducing equivalents to re-reduce the copper atoms at the active site following the catalytic chemistry [Chan, S. I., Wang, V. C.-C., Lai, J. C.-H., Yu, S. S.-F., Chen, P. P.-Y., Chen, K. H.-C., Chen, C.-L., and Chan, M. K. (2007) Redox potentiometry studies of particulate methane monooxygenase: Support for a trinuclear copper cluster active site, Angew. Chem., Int. Ed. 46, 1992-1994]. Since the aqueous-exposed domains of the 45 kDa subunit (PmoB) have been suggested to be the putative binding domains for the E-cluster copper ions, we have cloned and overexpressed in Escherichia coli the two aqueous-exposed subdomains toward the N- and C-termini of the subunit: the N-terminal subdomain (residues 54-178) and the C-terminal subdomain (residues 257-394 and 282-414). The recombinant C-terminal water-exposed subdomain is shown to behave like a Cu(I) sponge, taking up to ca. 10 Cu(I) ions cooperatively when cupric ions are added to the protein fragment in the presence of dithiothreitol or ascorbate. In addition, circular dichroism measurements reveal that the C-terminal subdomain folds into a beta-sheet structure in the presence of Cu(I). The propensity for the C-terminal subdomain to bind Cu(I) is consistent with the high redox potential(s) determined for the E-cluster copper ions in the pMMO. These properties of the E-clusters are in accordance with the function proposed for these copper ions in the turnover cycle of the enzyme.