EX VIVO STUDIES ON HOST RANGE AND TROPISM OF INFLUENZA A VIRUSES

Influenza A viruses (IAVs) are single stranded RNA viruses belonging to the Orthomyxoviridae family. These viruses exhibit high evolutionary rates and are present in a wide range of animal species. Host switching of IAVs is unpredictable and poses a great threat to human health. Therefore, understanding the underlying mechanisms driving such events is one of the current goals in influenza research. Among the different subtypes, H3N8 IAVs have shown a remarkable tendency to host species jumps. H3N8 equine influenza virus (EIV) is an avian-origin virus that circulated in horses for nearly 40 years before emerging in dogs in early 2000s. In the last decade H3N8 EIV has also been isolated from pigs, key hosts in influenza ecology, and other mammals. To understand the nature of host range determinants it is fundamental to study the dynamics of influenza pathogenesis at the site of infection. Respiratory explants constitute a suitable model to investigate virus replication in the target tissues of the host. This thesis aimed at thoroughly investigating the infection dynamics of host adapted and non-host adapted IAV infections in organ explants of pigs and horses. We first described the infection phenotype of host adapted viruses such as H3N2 swine influenza (SIV) and H3N8 equine influenza in tissues of their natural hosts to build a solid background knowledge on disease pathogenesis. Swine and equine explants were infected with SIV and EIV, respectively, and accurately monitored for viral replication and structural changes at the site of infection (chapters two and three). This extensive examination has added substantial information to previous literature and confirmed that swine and equine ex vivo systems support influenza replication and are suitable to study virus pathogenesis and at the same time observe the 3Rs ethos (Reduction, Replacement and Refinement). Next, we addressed our research interests on non-host adapted infections focusing on the H3N8 subtype. Enhancing the current knowledge on the role of evolution in the complex phenomenon of viral emergence is of utmost importance. To this end, in chapter four we compared the replication potential of phylogenetically distinct EIVs, each representing an evolutionary different period, in swine cell lines and respiratory explants. All tested EIVs replicated in cell lines whereas only the earliest EIV isolate (Uruguay/63) was able to infect swine explants with a marked tropism for the lower respiratory tract, demonstrating the presence of tissue-specific host barriers at the site of infection. The distinct phenotypes observed ex vivo support the view that evolutionary processes play important roles on host range and tropism of EIV. Nonetheless, when compared to SIV H3N2, EIV Uruguay/63 productively infected only a limited number of explants. H3N8 EIV has originated from the avian gene pool maintained in aquatic waterfowl. To further look into host range shifts of H3N8 IAVs, in chapter five we investigated the replication dynamics of H3N8 avian influenza viruses (AIVs) in equine tracheal explants. To give an ecologically plausible context to our experiments, we tested AIVs isolated from wild birds in Mongolia, a region densely populated with wild birds and horses. H3N8 AIVs infected tracheal explants, albeit to significantly lower levels and with a different infection phenotype compared to an EIV. This difference was evidenced as lower viral titres, absence of epithelial damage and difficult viral nucleoprotein detection in the infected tissue. These findings, coupled with serological evidence of AIV infections in horses in the field, suggest that introduction of viruses from the avian reservoir can occur and that further adaptive changes may be required for a successful establishment. Overall, by investigating the infection dynamics of IAVs in ex vivo cultures of the respiratory tract we have provided new insights into the different phenotypes displayed by host adapted and non-host adapted viruses at the site of infection. Our results support the hypothesis that viral evolution during long-term transmission of IAVs in host populations could result in dynamic changes in their host range. Such changes must be in line with ecological and epidemiological factors in order to allow the establishment of novel lineages in susceptible hosts. Finally, we have confirmed that organ explants can bridge important gaps between in vitro and in vivo experiments

Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer.

Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of preexisting subclones, remains unclear. In epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires Aurora kinase A (AURKA) activity. Nongenetic resistance through the activation of AURKA by its coactivator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in preclinical models. Treatment-induced activation of AURKA is associated with resistance to EGFR inhibitors in vitro, in vivo and in most individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a molecular path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA-driven residual disease and acquired resistance

The catalytic subunit of the system L1 amino acid transporter (S<i>lc7a5</i>) facilitates nutrient signalling in mouse skeletal muscle

The System L1-type amino acid transporter mediates transport of large neutral amino acids (LNAA) in many mammalian cell-types. LNAA such as leucine are required for full activation of the mTOR-S6K signalling pathway promoting protein synthesis and cell growth. The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98). We generated a floxed Slc7a5 mouse strain which, when crossed with mice expressing Cre driven by a global promoter, produced Slc7a5 heterozygous knockout (Slc7a5+/-) animals with no overt phenotype, although homozygous global knockout of Slc7a5 was embryonically lethal. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout (MS-Slc7a5-KO) mice were used to study the role of intracellular LNAA delivery by the SLC7A5 transporter for mTOR-S6K pathway activation in skeletal muscle. Activation of muscle mTOR-S6K (Thr389 phosphorylation) in vivo by intraperitoneal leucine injection was blunted in homozygous MS-Slc7a5-KO mice relative to wild-type animals. Dietary intake and growth rate were similar for MS-Slc7a5-KO mice and wild-type littermates fed for 10 weeks (to age 120 days) with diets containing 10%, 20% or 30% of protein. In MS-Slc7a5-KO mice, Leu and Ile concentrations in gastrocnemius muscle were reduced by ∼40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signalling in mice, although it appears non-essential (or is sufficiently compensated by e.g. SLC7A8 (LAT2)) for maintenance of normal muscle mass

Knockdown of hTERT and Treatment with BIBR1532 Inhibit Cell Proliferation and Invasion in Endometrial Cancer Cells

Telomerase activity and expression of the catalytic protein hTERT are associated with cell proliferation and advanced stage in endometrial cancer. Our objective was to evaluate the effect of inhibition of hTERT by siRNA and BIBR1532 on cell growth, apoptosis and invasion in endometrial cancer cells. Knockdown of hTERT or treatment of the cells with BIBR1532 decreased telomerase activity, inhibited cell proliferation, induced apoptosis, and reduced cell invasion in Ishikawa and ECC-1 cells. Either hTERT siRNA or BIBR1532 in combination with paclitaxel promoted a synergistic inhibitory effect on cell growth through induction of Annexin V expression and a remarkable reduction in cell invasion through reduction of protein expression of MMP9, MMP2, and MMP3. Increased telomerase activity and hTERT protein expression by transfections enhanced the protein expression of MMPs and increased the cell invasion ability. BIBR1532 significantly antagonized cell invasion induced by increased hTERT expression. These findings suggest that telomerase and hTERT facilitate cell invasion via MMP family in human endometrial cancer cells

Granular cell tumors of the urinary bladder

BACKGROUND: Granular cell tumors (GCTs) are extremely rare lesions of the urinary bladder with only nine cases being reported in world literature of which one was malignant. Generally believed to be of neural origin based on histochemical, immunohistochemical, and ultrastructural studies; they mostly follow a clinically benign course but are commonly mistaken for malignant tumors since they are solid looking, ulcerated tumors with ill-defined margins. MATERIALS AND METHODS: We herein report two cases of GCTs, one benign and one malignant, presenting with gross hematuria in a 14- and a 47-year-old female, respectively. RESULTS: Histopathology revealed characteristic GCTs with positive immunostaining for neural marker (S-100) and negative immunostaining for epithelial (cytokeratin, Cam 5.2, AE/A13), neuroendocrine (neuron specific enolase, chromogranin A, and synaptophysin) and sarcoma (desmin, vimentin) markers. The benign tumor was successfully managed conservatively with transurethral resection alone while for the malignant tumor, radical cystectomy, hysterectomy with bilateral salpingo-oophorectomy, anterior vaginectomy, plus lymph node dissection was done. Both cases show long-term disease free survival. CONCLUSION: We recommend careful pathologic assessment for establishing the appropriate diagnosis and either a conservative or aggressive surgical treatment for benign or localized malignant GCT of the urinary bladder, respectively

Do salivary bypass tubes lower the incidence of pharyngocutaneous fistula following total laryngectomy? A retrospective analysis of predictive factors using multivariate analysis

Salivary bypass tubes (SBT) are increasingly used to prevent pharyngocutaneous fistula (PCF) following laryngectomy and pharyngolaryngectomy. There is minimal evidence as to their efficacy and literature is limited. The aim of the study was to determine if SBT prevent PCF. The study was a multicentre retrospective case control series (level of evidence 3b). Patients who underwent laryngectomy or pharyngolaryngectomy for cancer or following cancer treatment between 2011 and 2014 were included in the study. The primary outcome was development of a PCF. Other variables recorded were age, sex, prior radiotherapy or chemoradiotherapy, prior tracheostomy, type of procedure, concurrent neck dissection, use of flap reconstruction, use of prophylactic antibiotics, the suture material used for the anastomosis, tumour T stage, histological margins, day one post-operative haemoglobin and whether a salivary bypass tube was used. Univariate and multivariate analysis were performed. A total of 199 patients were included and 24 received salivary bypass tubes. Fistula rates were 8.3% in the SBT group (2/24) and 24.6% in the control group (43/175). This was not statistically significant on univariate (p value 0.115) or multivariate analysis (p value 0.076). In addition, no other co-variables were found to be significant. No group has proven a benefit of salivary bypass tubes on multivariate analysis. The study was limited by a small case group, variations in tube duration and subjects given a tube may have been identified as high risk of fistula. Further prospective studies are warranted prior to recommendation of salivary bypass tubes following laryngectomy

The Transcriptome Analysis of Strongyloides stercoralis L3i Larvae Reveals Targets for Intervention in a Neglected Disease

BackgroundStrongyloidiasis is one of the most neglected diseases distributed worldwide with endemic areas in developed countries, where chronic infections are life threatening. Despite its impact, very little is known about the molecular biology of the parasite involved and its interplay with its hosts. Next generation sequencing technologies now provide unique opportunities to rapidly address these questions.Principal FindingsHere we present the first transcriptome of the third larval stage of S. stercoralis using 454 sequencing coupled with semi-automated bioinformatic analyses. 253,266 raw sequence reads were assembled into 11,250 contiguous sequences, most of which were novel. 8037 putative proteins were characterized based on homology, gene ontology and/or biochemical pathways. Comparison of the transcriptome of S. strongyloides with those of other nematodes, including S. ratti, revealed similarities in transcription of molecules inferred to have key roles in parasite-host interactions. Enzymatic proteins, like kinases and proteases, were abundant. 1213 putative excretory/secretory proteins were compiled using a new pipeline which included non-classical secretory proteins. Potential drug targets were also identified.ConclusionsOverall, the present dataset should provide a solid foundation for future fundamental genomic, proteomic and metabolomic explorations of S. stercoralis, as well as a basis for applied outcomes, such as the development of novel methods of intervention against this neglected parasite