Harnessing endogenous peptide compounds as potential therapeutics for severe influenza.

BACKGROUND: Excessive pulmonary inflammation and damage are characteristic features of severe influenza virus infections. LAT8881 is a synthetic, 16 amino acid cyclic peptide form of a naturally occurring C-terminal fragment of human growth hormone with therapeutic efficacy against influenza. Shorter, linear peptides are typically easier to manufacture and formulate for delivery than larger cyclic peptides. A 6 amino acid linear peptide fragment of LAT8881, LAT9997, was investigated as a potential influenza therapy. METHODS: LAT9997 was evaluated for its potential to limit disease in a preclinical mouse model of severe influenza infection. RESULTS: Intranasal treatment of mice with either LAT8881 or LAT9997 from day 1 following influenza infection significantly improved survival outcomes. Initiating LAT9997 treatment at the onset of severe disease, also significantly improved disease severity. Greater disease resistance in LAT9997-treated mice correlated with reduced lung immunopathology, damage markers, vascular leak, and epithelial cell death. Treatment reduced viral loads, cytokines, and neutrophil infiltration in the airways, yet maintained protective alveolar macrophages in a dose-dependent manner. Sequential trimming of N- and C-terminal amino acids from LAT9997 revealed a structure-activity relationship. CONCLUSIONS: These findings provide preclinical evidence that therapeutic LAT9997 treatment limits viral burden and characteristic features of severe influenza, including hyperinflammation and lung damage.

Gasdermin D promotes hyperinflammation and immunopathology during severe influenza A virus infection.

Excessive inflammation and tissue damage during severe influenza A virus (IAV) infection can lead to the development of fatal pulmonary disease. Pyroptosis is a lytic and pro-inflammatory form of cell death executed by the pore-forming protein gasdermin D (GSDMD). In this study, we investigated a potential role for GSDMD in promoting the development of severe IAV disease. IAV infection resulted in cleavage of GSDMD in vivo and in vitro in lung epithelial cells. Mice genetically deficient in GSDMD (Gsdmd-/-) developed less severe IAV disease than wildtype mice and displayed improved survival outcomes. GSDMD deficiency significantly reduced neutrophil infiltration into the airways as well as the levels of pro-inflammatory cytokines TNF, IL-6, MCP-1, and IL-1α and neutrophil-attracting chemokines CXCL1 and CXCL2. In contrast, IL-1ß and IL-18 responses were not largely impacted by GSDMD deficiency. In addition, Gsdmd-/- mice displayed significantly improved influenza disease resistance with reduced viral burden and less severe pulmonary pathology, including decreased epithelial damage and cell death. These findings indicate a major role for GSDMD in promoting damaging inflammation and the development of severe IAV disease.

Naturally derived cytokine peptides limit virus replication and severe disease during influenza A virus infection.

Objectives: Novel host-targeted therapeutics could treat severe influenza A virus (IAV) infections, with reduced risk of drug resistance. LAT8881 is a synthetic form of the naturally occurring C-terminal fragment of human growth hormone. Acting independently of the growth hormone receptor, it can reduce inflammation-induced damage and promote tissue repair in an animal model of osteoarthritis. LAT8881 has been assessed in clinical trials for the treatment of obesity and neuropathy and has an excellent safety profile. We investigated the potential for LAT8881, its metabolite LAT9991F and LAT7771 derived from prolactin, a growth hormone structural homologue, to treat severe IAV infection. Methods: LAT8881, LAT9991F and LAT7771 were evaluated for their effects on cell viability and IAV replication in vitro, as well as their potential to limit disease in a preclinical mouse model of severe IAV infection. Results: In vitro LAT8881 treatment enhanced cell viability, particularly in the presence of cytotoxic stress, which was countered by siRNA inhibition of host lanthionine synthetase C-like proteins. Daily intranasal treatment of mice with LAT8881 or LAT9991F, but not LAT7771, from day 1 postinfection significantly improved influenza disease resistance, which was associated with reduced infectious viral loads, reduced pro-inflammatory cytokines and increased abundance of protective alveolar macrophages. LAT8881 treatment in combination with the antiviral oseltamivir phosphate led to more pronounced reduction in markers of disease severity than treatment with either compound alone. Conclusion: These studies provide the first evidence identifying LAT8881 and LAT9991F as novel host-protective therapies that improve survival, limit viral replication, reduce local inflammation and curtail tissue damage during severe IAV infection. Evaluation of LAT8881 and LAT9991F in other infectious and inflammatory conditions of the airways is warranted.

Blockade of the protease ADAM17 ameliorates experimental pancreatitis.

Acute and chronic pancreatitis, the latter associated with fibrosis, are multifactorial inflammatory disorders and leading causes of gastrointestinal disease-related hospitalization. Despite the global health burden of pancreatitis, currently, there are no effective therapeutic agents. In this regard, the protease A Disintegrin And Metalloproteinase 17 (ADAM17) mediates inflammatory responses through shedding of bioactive inflammatory cytokines and mediators, including tumor necrosis factor α (TNFα) and the soluble interleukin (IL)-6 receptor (sIL-6R), the latter of which drives proinflammatory IL-6 trans-signaling. However, the role of ADAM17 in pancreatitis is unclear. To address this, Adam17ex/ex mice-which are homozygous for the hypomorphic Adam17ex allele resulting in marked reduction in ADAM17 expression-and their wild-type (WT) littermates were exposed to the cerulein-induced acute pancreatitis model, and acute (1-wk) and chronic (20-wk) pancreatitis models induced by the cigarette smoke carcinogen nicotine-derived nitrosamine ketone (NNK). Our data reveal that ADAM17 expression was up-regulated in pancreatic tissues of animal models of pancreatitis. Moreover, the genetic (Adam17ex/ex mice) and therapeutic (ADAM17 prodomain inhibitor [A17pro]) targeting of ADAM17 ameliorated experimental pancreatitis, which was associated with a reduction in the IL-6 trans-signaling/STAT3 axis. This led to reduced inflammatory cell infiltration, including T cells and neutrophils, as well as necrosis and fibrosis in the pancreas. Furthermore, up-regulation of the ADAM17/IL-6 trans-signaling/STAT3 axis was a feature of pancreatitis patients. Collectively, our findings indicate that the ADAM17 protease plays a pivotal role in the pathogenesis of pancreatitis, which could pave the way for devising novel therapeutic options to be deployed against this disease.

Oncogenic dependency on STAT3 serine phosphorylation in KRAS mutant lung cancer.

The oncogenic potential of the latent transcription factor signal transducer and activator of transcription (STAT)3 in many human cancers, including lung cancer, has been largely attributed to its nuclear activity as a tyrosine-phosphorylated (pY705 site) transcription factor. By contrast, an alternate mitochondrial pool of serine phosphorylated (pS727 site) STAT3 has been shown to promote tumourigenesis by regulating metabolic processes, although this has been reported in only a restricted number of mutant RAS-addicted neoplasms. Therefore, the involvement of STAT3 serine phosphorylation in the pathogenesis of most cancer types, including mutant KRAS lung adenocarcinoma (LAC), is unknown. Here, we demonstrate that LAC is suppressed in oncogenic KrasG12D-driven mouse models engineered for pS727-STAT3 deficiency. The proliferative potential of the transformed KrasG12D lung epithelium, and mutant KRAS human LAC cells, was significantly reduced upon pS727-STAT3 deficiency. Notably, we uncover the multifaceted capacity of constitutive pS727-STAT3 to metabolically reprogramme LAC cells towards a hyper-proliferative state by regulating nuclear and mitochondrial (mt) gene transcription, the latter via the mtDNA transcription factor, TFAM. Collectively, our findings reveal an obligate requirement for the transcriptional activity of pS727-STAT3 in mutant KRAS-driven LAC with potential to guide future therapeutic targeting approaches.

ADAM17 Deficiency Protects against Pulmonary Emphysema.

Pulmonary emphysema is the major debilitating component of chronic obstructive pulmonary disease (COPD), which is a leading cause of morbidity and mortality worldwide. The ADAM17 (A disintegrin and metalloproteinase 17) protease mediates inflammation via ectodomain shedding of numerous proinflammatory cytokines, cytokine receptors, and adhesion molecules; however, its role in the pathogenesis of emphysema and COPD is poorly understood. This study aims to define the role of the protease ADAM17 in the pathogenesis of pulmonary emphysema. ADAM17 protein expression and activation was investigated in lung biopsies from patients with emphysema, as well as lungs of the emphysematous gp130F/F mouse model and an acute (4 d) cigarette smoke (CS)-induced lung pathology model. The Adam17ex/ex mice, which display significantly reduced global ADAM17 expression, were coupled with emphysema-prone gp130F/F mice to produce gp130F/F:Adam17ex/ex. Both Adam17ex/ex and wild-type mice were subjected to acute CS exposure. Histological, immunohistochemical, immunofluorescence, and molecular analyses as well as lung function tests were performed to assess pulmonary emphysema, inflammation, and alveolar cell apoptosis. ADAM17 was hyperphosphorylated in the lungs of patients with emphysema and also in emphysematous gp130F/F and CS-exposed mice. ADAM17 deficiency ameliorated the development of pulmonary emphysema in gp130F/F mice by suppressing elevated alveolar cell apoptosis. In addition, genetic blockade of ADAM17 protected mice from CS-induced pulmonary inflammation and alveolar cell apoptosis. Our study places the protease ADAM17 as a central molecular switch implicated in the development of pulmonary emphysema, which paves the way for using ADAM17 inhibitors as potential therapeutic agents to treat COPD and emphysema.

The effects of acute alcohol withdrawal on sleep.

OBJECTIVES: Although sleep disturbances are prominent during alcohol withdrawal, less is known about the specific components of sleep that are disturbed prior to and during acute detoxification. This study aimed to determine whether specific sleep components are affected prior to and during acute detoxification and their relationship to psychological distress. METHODS: Twenty-nine participants were recruited from a residential detoxification service in Melbourne, Australia, and completed both subjective methods of sleep and distress, in addition to wearing an actigraphy device. RESULTS: Daytime dysfunction, sleep quality, and sleep disturbances were the components that were most disturbed in the month prior to admission, and poor sleep efficiency was detected during acute withdrawal using actigraphy. A significant association was found between sleep and psychological distress in this group. CONCLUSIONS: Specific disturbances in sleep are experienced prior to and during acute alcohol withdrawal, suggesting that tailored interventions may be effective in the treatment of sleep deficits during these periods.

Novel impedimetric immunosensor for the detection and quantitation of Adenovirus using reduced antibody fragments immobilized onto a conducting copolymer surface.

The number of Adenovirus (Ad) infections detected in immunocompromised people has increased due to the number of patients receiving transplants, as well as the HIV pandemic. Ads cause life-threatening diseases specific to the infected organs of immunocompromised hosts, with discontinuation of immunosuppressive agents necessary to prevent morbidity. The methodology in this paper has been employed to develop a novel impedimetric based assay platform to detect and quantify human Ads, which is comparable in performance to current methods, such as ELISA and PCR, but is also less expensive and faster. Novel immunosensors have been fabricated using polyclonal antibodies raised against a human Ad (Ad5) capsid protein, which were selectively cleaved into antibody fragments by 2-mercaptoethylamine. The fragments were immobilized onto a functionalized conducting copolymer matrix comprising polyaniline and 2-aminobenzylamine. Fully fabricated sensors were incubated with two immunologically distinct serotypes of Ad, Ad5 and Ad3, with between 10 and 10(12)virus particles/mL prior to sensor interrogation. Electrochemical impedance spectroscopy was used to measure the charge transfer resistance of the sensors over a range of frequencies from 25 kHz to 0.1 Hz. Our data demonstrate that the immunosensors specifically detect, and differentiate between, closely related human Ad serotypes with a limit of detection of 10(3)virus particles/mL. In addition, atomic force microscopy was applied to study the sensor surface nanostructure. Future work looks to test virus containing clinical samples but this could be a viable and valuable alternative for point-of-care virus detection and quantification.

PTPan--overcoming memory limitations in oligonucleotide string matching for primer/probe design.

MOTIVATION: Nucleic acid diagnostics has high demands for non-heuristic exact and approximate oligonucleotide string matching concerning in silico primer/probe design in huge nucleic acid sequence collections. Unfortunately, public sequence repositories grow much faster than computer hardware performance and main memory capacity do. This growth imposes severe problems on existing oligonucleotide primer/probe design applications necessitating new approaches based on space-efficient indexing structures. RESULTS: We developed PTPan (spoken Peter Pan, 'PT' is for Position Tree, the earlier name of suffix trees), a space-efficient indexing structure for approximate oligonucleotide string matching in nucleic acid sequence data. Based on suffix trees, it combines partitioning, truncation and a new suffix tree stream compression to deal with large amounts of aligned and unaligned data. PTPan operates efficiently in main memory and on secondary storage, balancing between memory consumption and runtime during construction and application. Based on PTPan, applications supporting similarity search and primer/probe design have been implemented, namely FindFamily, ProbeMatch and ProbeDesign. All three use a weighted Levenshtein distance metric for approximative queries to find and rate matches with indels as well as substitutions. We integrated PTPan in the worldwide used software package ARB to demonstrate usability and performance. Comparing PTPan and the original ARB index for the very large ssu-rRNA database SILVA, we recognized a shorter construction time, extended functionality and dramatically reduced memory requirements at the price of expanded, but very reasonable query times. PTPan enables indexing of huge nucleic acid sequence collections at reasonable application response times. Not being limited by main memory, PTPan constitutes a major advancement regarding rapid oligonucleotide string matching in primer/probe design now and in the future facing the enormous growth of molecular sequence data. AVAILABILITY: Supplementary Material, PTPan stand-alone library and ARB-PTPan binary on http://ptpan.lrr.in.tum.de/. CONTACT: meierh@in.tum.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing.

The successful construction of an immunosensor depends on having an effective procedure for immobilising the bio-recognition element to the transducer surface. In the present study, an amino-terminated 4-aminothiophenol (ATP) self-assembled monolayer (SAM) was modified with heterobifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate to couple reduced anti-myoglobin half-antibody fragments. The disulphide groups present in the hinge region of IgG molecules were selectively cleaved by 2-mercaptoethylamine to produce reduced half-antibody fragments with free sulphydryl groups. The maleimide terminated 4-ATP SAM modified surface was coupled to these reduced antibody fragments to produce highly oriented immobilization of the half-antibody via its Fc domain and to allow free access to the Fv bindings sites. This represents an improvement by comparison with biotin/avidin mediated IgG attachment which is essentially randomly oriented. Functional immunosensors were able to detect myoglobin in both phosphate buffered saline and whole serum over the range of concentrations from 10(-13)M to 10(-6)M, and order of magnitude better than avidin/biotin linked immunosensors. In addition, atomic force microscopy (AFM) was carried out to elucidate the nanotopology of the immunosensor surface at different stages of fabrication; the images demonstrate that half antibodies bind as described and show structural changes on subsequent antigen binding.

Simultaneous neutron scattering and Raman scattering.

The capability to make simultaneous neutron and Raman scattering measurements at temperatures between 1.5 and 450 K has been developed. The samples to be investigated are attached to one end of a custom-made center-stick suitable for insertion into a 100 mm-bore cryostat. The other end of the center-stick is fiber-optically coupled to a Renishaw in Via Raman spectrometer incorporating a 300 mW Toptica 785 nm wavelength stabilized diode laser. The final path for the laser beam is approximately 1.3 m in vacuo within the center-stick followed by a focusing lens close to the sample. Raman scattering measurements with a resolution of 1 to 4 cm(-1) can be made over a wide range (100-3200 cm(-1)) at the same time as a variety of different types of neutron scattering measurements. In this work we highlight the use of inelastic neutron scattering and neutron diffraction in conjunction with the Raman for studies of the globular protein lysozyme.