Detection of Cell-Dissociated Non-Typeable Haemophilus influenzae in the Airways of Patients with Chronic Obstructive Pulmonary Disease.

Background: Non-typeable Haemophilus influenzae (NTHi) is the most commonly found pathogen in the lower respiratory airways of patients with COPD. NTHi is predominantly regarded as an intracellular pathogen; however, like most pathogens, it can exist and co-exist in two broad forms: cell-associated (intracellularly or adhered to cells) or cell-dissociated (biofilm dispersed or planktonic). We sought to investigate if cell-dissociated NTHi can be detected from the sputum of COPD patients and assess this relationship to disease severity and airway inflammation. Methods: DNA was extracted from the sputum plug and cell-free supernatant to quantify absolute (cell-associated and cell-dissociated NTHi) and cell-dissociated NTHi, respectively, from 87 COPD subjects attending an observational longitudinal COPD exacerbation study. NTHi was quantified using TaqMan hydrolysis probes, targeting the OMP P6 gene using qPCR. Results: At stable state cell-dissociated NTHi was detected 56% of subjects with a median (IQR) of 9.95x102 gene copies (1.26x102 to 1.90x104). Cell-dissociated NTHi correlated with absolute NTHi levels (r=0.34, p<0.01) but not airway inflammation or spirometry at stable state. At exacerbation, cell-dissociated NTHi correlated with lung function (FEV1 r=0.629, p=0.005; FEV1%predicted r=0.564, p=0.015; FVC r=0.476 p=0.046) and sputum neutrophilic inflammation (% neutrophils r=0.688, p=0.002; total neutrophils r=0.518, p=0.028). Conclusion: In patients with COPD, NTHi can exist in both cell-associated and cell-dissociated forms. Cell-dissociated NTHi is associated with neutrophilic airway inflammation during exacerbations of COPD and may be a driving factor in worsening lung function during these episodes.

Identification of molecular targets for the targeted treatment of gastric cancer using dasatinib.

Gastric cancer (GC) remains the third leading cause of cancer-related death despite several improvements in targeted therapy. There is therefore an urgent need to investigate new treatment strategies, including the identification of novel biomarkers for patient stratification. In this study, we evaluated the effect of FDA-approved kinase inhibitors on GC. Through a combination of cell growth, migration and invasion assays, we identified dasatinib as an efficient inhibitor of GC proliferation. Mass-spectrometry-based selectivity profiling and subsequent knockdown experiments identified members of the SRC family of kinases including SRC, FRK, LYN and YES, as well as other kinases such as DDR1, ABL2, SIK2, RIPK2, EPHA2, and EPHB2 as dasatinib targets. The expression levels of the identified kinases were investigated on RNA and protein level in 200 classified tumor samples from patients, who had undergone gastrectomy, but had received no treatment. Levels of FRK, DDR1 and SRC expression on both mRNA and protein level were significantly higher in metastatic patient samples regardless of the tumor stage, while expression levels of SIK2 correlated with tumor size. Collectively, our data suggest dasatinib for treatment of GC based on its unique property, inhibiting a small number of key kinases (SRC, FRK, DDR1 and SIK2), highly expressed in GC patients.

Bi-directional signaling by membrane-bound KitL induces proliferation and coordinates thymic endothelial cell and thymocyte expansion.

The ligand for the c-Kit receptor, KitL, exists as a membrane-associated (mKitL) and a soluble form (sKitL). KitL functions outside c-Kit activation have not been identified. We show that co-culture of c-Kit- and mKitL-expressing NIH3T3 cells results in signaling through mKitL: c-Kit-bound mKitL recruits calcium-modulating cyclophilin ligand (CAML) to selectively activate Akt, leading to CREB phosphorylation, mTOR pathway activation, and increased cell proliferation. Activation of mKitL in thymic vascular endothelial cells (VECs) induces mKitL- and Akt-dependent proliferation, and genetic ablation of mKitL in thymic VECs blocks their c-Kit responsiveness and proliferation during neonatal thymic expansion. Therefore, mKitL-c-Kit form a bi-directional signaling complex that acts in the developing thymus to coordinate thymic VEC and early thymic progenitor (ETP) expansion by simultaneously promoting ETP survival and VEC proliferation. This mechanism may be relevant to both normal tissues and malignant tumors that depend on KitL-c-Kit signaling for their proliferation.

Benzimidazoles Promote Anti-TNF Mediated Induction of Regulatory Macrophages and Enhance Therapeutic Efficacy in a Murine Model.

BACKGROUND AND AIMS: Regulatory macrophages play a critical role in tissue repair, and we have previously shown that anti-tumour necrosis factor [TNF] antibodies induce these macrophages in vitro and in vivo in IBD patients. The induction of regulatory macrophages can be potentiated using the combination of anti-TNF and thiopurines, consistent with the enhanced efficacy of this combination therapy described in clinical trials. As thiopurines are unfortunately associated with significant side effects, we here aimed to identify alternatives for combination therapy with anti-TNF, using the macrophage induction model as a screening tool. METHODS: Mixed lymphocyte reactions were treated with anti-TNF and a library of 1600 drug compounds. Induction of CD14+CD206+ macrophages was analysed by flow cytometry. Positive hits were validated in vitro and in the T cell transfer model of colitis. RESULTS: Among the 98 compounds potentiating the induction of regulatory macrophages by anti-TNF were six benzimidazoles, including albendazole. Albendazole treatment in the presence of anti-TNF resulted in alterations in the tubulin skeleton and signalling though AMPK, which was required for the enhanced induction. Combination therapy also increased expression levels of the immunoregulatory cytokine IL-10. In vivo, albendazole plus anti-TNF combination therapy was superior to monotherapy in a model of colitis, in terms of both induction of regulatory macrophages and improvement of clinical symptoms. CONCLUSIONS: Albendazole enhances the induction of regulatory macrophages by anti-TNF and potentiates clinical efficacy in murine colitis. Given its favourable safety profile, these data indicate that the repurposing of albendazole may be a novel option for anti-TNF combination therapy in IBD.

In Vitro-Pooled shRNA Screening to Identify Determinants of Radiosensitivity.

Short hairpin RNA (shRNA)-pooled screening is a valuable and cost-effective tool for assaying the contribution of individual genes to cell viability and proliferation on a genomic scale. Here we describe the key considerations for the design and execution of a pooled shRNA screen to identify determinants of radiosensitivity.

Systematic Functional Characterization of Candidate Causal Genes for Type 2 Diabetes Risk Variants.

Most genetic association signals for type 2 diabetes risk are located in noncoding regions of the genome, hindering translation into molecular mechanisms. Physiological studies have shown a majority of disease-associated variants to exert their effects through pancreatic islet dysfunction. Systematically characterizing the role of regional transcripts in ß-cell function could identify the underlying disease-causing genes, but large-scale studies in human cellular models have previously been impractical. We developed a robust and scalable strategy based on arrayed gene silencing in the human ß-cell line EndoC-ßH1. In a screen of 300 positional candidates selected from 75 type 2 diabetes regions, each gene was assayed for effects on multiple disease-relevant phenotypes, including insulin secretion and cellular proliferation. We identified a total of 45 genes involved in ß-cell function, pointing to possible causal mechanisms at 37 disease-associated loci. The results showed a strong enrichment for genes implicated in monogenic diabetes. Selected effects were validated in a follow-up study, including several genes (ARL15, ZMIZ1, and THADA) with previously unknown or poorly described roles in ß-cell biology. We have demonstrated the feasibility of systematic functional screening in a human ß-cell model and successfully prioritized plausible disease-causing genes at more than half of the regions investigated.

BET inhibition as a new strategy for the treatment of gastric cancer.

Gastric cancer is one of the most common malignancies and a leading cause of cancer death worldwide. The prognosis of stomach cancer is generally poor as this cancer is not very sensitive to commonly used chemotherapies. Epigenetic modifications play a key role in gastric cancer and contribute to the development and progression of this malignancy. In order to explore new treatment options in this target area we have screened a library of epigenetic inhibitors against gastric cancer cell lines and identified inhibitors for the BET family of bromodomains as potent inhibitors of gastric cancer cell proliferations. Here we show that both the pan-BET inhibitor (+)-JQ1 as well as a newly developed specific isoxazole inhibitor, PNZ5, showed potent inhibition of gastric cancer cell growth. Intriguingly, we found differences in the antiproliferative response between gastric cancer cells tested derived from Brazilian patients as compared to those from Asian patients, the latter being largely resistant to BET inhibition. As BET inhibitors are entering clinical trials these findings provide the first starting point for future therapies targeting gastric cancer.

Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity.

In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.

Systematic analysis of the IgG antibody immune response against varicella zoster virus (VZV) using a self-assembled protein microarray.

Varicella zoster virus (VZV) is a human herpesvirus encoding at least 69 distinct viral proteins which causes chickenpox after primary infection and shingles during reactivation and which is particularly important in pregnancy and immunocompromised patients. Current serodiagnostic tests are either based on whole cell lysates or glycoprotein preparations. In order to investigate the humoral immune response to VZV infection or vaccination in more detail, and to improve the currently available diagnostic assays, we developed a nucleic acid programmable protein array (NAPPA) containing all 69 VZV proteins and performed a detailed analysis of 68 sera from individuals with either no, a previous or an acute VZV infection. In addition to the known reactive glycoprotein antigens (ORF 5, ORF 14, ORF 31, ORF 37, ORF 68), we discovered IgG antibodies against a variety of other membrane (ORF 2, ORF 24), capsid (ORF 20, ORF 23, ORF 43) and tegument (ORF 53, ORF 9, ORF 11) proteins, as well as other proteins involved in virus replication and assembly (ORF 25, ORF 26, ORF 28) and the transactivator proteins ORF 12, ORF 62 and ORF 63. All of these antigens were only reactive in a subset of VZV-positive individuals. A subset of the newly identified VZV antigens was validated by western blot analysis. Using these seroreactive new VZV antigens, more sensitive assays and tests distinguishing between different clinical entities may be developed.