Fasciola hepatica vaccine: We may not be there yet but we're on the right road

Major advances have been made in identifying potential vaccine molecules for the control of fasciolosis in livestock but we have yet to reach the level of efficacy required for commercialisation. The pathogenesis of fasciolosis is associated with liver damage that is inflicted by migrating and feeding immature flukes as well as host inflammatory immune responses to parasite-secreted molecules and tissue damage alarm signals. Immune suppression/modulation by the parasites prevents the development of protective immune responses as evidenced by the lack of immunity observed in naturally and experimentally infected animals. In our opinion, future efforts need to focus on understanding how parasites invade and penetrate the tissues of their hosts and how they potentiate and control the ensuing immune responses, particularly in the first days of infection. Emerging 'omics' data employed in an unbiased approach are helping us understand liver fluke biology and, in parallel with new immunological data, to identify molecules that are essential to parasite development and accessible to vaccine-induced immune responses.

Animal models of diabetic retinopathy

Diabetic retinopathy (DR) is a major cause of visual impairment worldwide. The precise pathogenesis of this diabetic complication remains ill-defined and this is reflected in the limited options for preventing development and progression of this disease. The value of animal models to understand and treat human disease is well recognised and this chapter focuses on the range of in vivo model systems that are available for studying DR. These models have been developed over many decades and utilised to aid our understanding of what causes DR, about how microvascular and neural lesions develop and to provide evidence for key cellular and molecular mechanisms that drive this pathology. A wide range of animal models of DR are currently available, each with advantages and disadvantages that need to be understood and evaluated for their scientific and clinical value. As transgenic and imaging technology improves, more models will be developed and they will continue to play a critical role in the development of new therapeutic approaches to DR by providing robust, preclinical evidence prior to clinical trial.

Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

The impact of climate change on existing and emerging microbial threats across the food chain: an island of Ireland perspective

The Agri-Food and aquaculture industries are vital to the economy of the island of Ireland with a gross annual output that is expected to double in the future. Identifying and understanding the potential influences of the anticipated climate variables on microorganisms that cause foodborne diseases, and their impact on these local industries, are essential. Investigating and monitoring foodborne pathogens and factors that influence their growth, transmission, pathogenesis and survival will facilitate assessment of the stability, security and vulnerability of the continuously evolving and increasing complex local food supply chain.

Microbial Communities Associated with Healthy and White Syndrome-Affected Echinopora lamellosa in Aquaria and Experimental Treatment with the Antibiotic Ampicillin

Prokaryotic and ciliate communities of healthy and aquarium White Syndrome (WS)-affected coral fragments were screened using denaturing gradient gel electrophoresis (DGGE). A significant difference (R = 0.907, p < 0.001) in 16S rRNA prokaryotic diversity was found between healthy (H), sloughed tissue (ST), WS-affected (WSU) and antibiotic treated (WST) samples. Although 3 Vibrio spp were found inWS-affected samples, two of these species were eliminated following ampicillin treatment, yet lesions continued to advance, suggesting they play a minor or secondary role in the pathogenesis. The third Vibrio sp increased slightly in relative abundance in diseased samples and was abundant in non-diseased samples. Interestingly, a Tenacibaculum sp showed the greatest increase in relative abundance between healthy and WS-affected samples, demonstrating consistently high abundance across all WS-affected and treated samples, suggesting Tenacibaculum sp could be a more likely candidate for pathogenesis in this instance. In contrast to previous studies bacterial abundance did not vary significantly (ANOVA, F2, 6 = 1.000, p = 0.422) between H, ST, WSU or WST. Antimicrobial activity (assessed on Vibrio harveyi cultures) was limited in both H and WSU samples (8.1% ±8.2 and 8.0% ±2.5, respectively) and did not differ significantly (Kruskal-Wallis, χ2 (2) = 3.842, p = 0.146). A Philaster sp, a Cohnilembus sp and a Pseudokeronopsis sp. were present in all WS-affected samples, but not in healthy samples. The exact role of ciliates in WS is yet to be determined, but it is proposed that they are at least responsible for the neat lesion boundary observed in the disease.

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of PI 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella containing vacuolae (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not colocalize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the downregulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation which may contribute to Klebsiella pathogenesis.

Evaluating early administration of the hydroxymethylglutaryl-CoA reductase inhibitor simvastatin in the prevention and treatment of delirium in critically ill ventilated patients (MoDUS trial): Study protocol for a randomized controlled trial

Background: The incidence of delirium in ventilated patients is estimated at up to 82%, and it is associated with longer intensive care and hospital stays, and long-term cognitive impairment and mortality. The pathophysiology of delirium has been linked with inflammation and neuronal apoptosis. Simvastatin has pleiotropic properties; it penetrates the brain and, as well as reducing cholesterol, reduces inflammation when used at clinically relevant doses over the short term. This is a single centre randomised, controlled trial which aims to test the hypothesis that treatment with simvastatin will modify delirium incidence and outcomes. 

Methods/Design: The ongoing study will include 142 adults admitted to the Watford General Hospital Intensive Care Unit who require mechanical ventilation in the first 72 hours of admission. The primary outcome is the number of delirium- and coma-free days in the first 14 days. Secondary outcomes include incidence of delirium, delirium- and coma-free days in the first 28 days, days in delirium and in coma at 14 and 28 days, number of ventilator-free days at 28 days, length of critical care and hospital stay, mortality, cognitive decline and healthcare resource use. Informed consent will be taken from patient's consultee before randomisation to receive either simvastatin (80 mg) or placebo once daily. Daily data will be recorded until day 28 after randomisation or until discharge from the ICU if sooner. Surviving patients will be followed up on at six months from discharge. Plasma and urine samples will be taken to investigate the biological effect of simvastatin on systemic markers of inflammation, as related to the number of delirium- and coma-free days, and the potential of cholinesterase activity and beta-amyloid as predictors of the risk of delirium and long-term cognitive impairment. 

Discussion: This trial will test the efficacy of simvastatin on reducing delirium in the critically ill. If patients receiving the statin show a reduced number of days in delirium compared with the placebo group, the inflammatory theory implicated in the pathogenesis of delirium will be strengthened. 

Comparative analysis of Klebsiella pneumoniae genomes identifies a phospholipase D family protein as a novel virulence factor

BACKGROUND: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044.

RESULTS: In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism.

CONCLUSIONS: Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae.

Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells

The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34 + cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript andor exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicingprocessing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expressionsplicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34 + cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processespathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months' streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.

BRCA1--conductor of the breast stem cell orchestra:the role of BRCA1 in mammary gland development and identification of cell of origin of BRCA1 mutant breast cancer

Breast cancer treatment has been increasingly successful over the last 20 years due in large part to targeted therapies directed against different subtypes. However, basal-like breast cancers still represent a considerable challenge to clinicians and scientists alike since the pathogenesis underlying the disease and the target cell for transformation of this subtype is still undetermined. The considerable similarities between basal-like and BRCA1 mutant breast cancers led to the hypothesis that these cancers arise from transformation of a basal cell within the normal breast epithelium through BRCA1 dysfunction. Recently, however, a number of studies have called this hypothesis into question. This review summarises the initial findings which implicated the basal cell as the cell of origin of BRCA1 related basal-like breast cancers, as well as the more recent data which identifies the luminal progenitor cells as the likely target of transformation. We compare a number of key studies in this area and identify the differences that could explain some of the contradictory findings. In addition, we highlight the role of BRCA1 in breast cell differentiation and lineage determination by reviewing recent findings in the field and our own observations suggesting a role for BRCA1 in stem cell regulation through activation of the p63 and Notch pathways. We hope that through an increased understanding of the BRCA1 role in breast differentiation and the identification of the cell(s) of origin we can improve treatment options for both BRCA1 mutant and basal-like breast cancer subgroups.

Deregulation of genes related to iron and mitochondrial metabolism in refractory anemia with ring sideroblasts

The presence of SF3B1 gene mutations is a hallmark of refractory anemia with ring sideroblasts (RARS). However, the mechanisms responsible for iron accumulation that characterize the Myelodysplastic Syndrome with ring sideroblasts (MDS-RS) are not completely understood. In order to gain insight in the molecular basis of MDS-RS, an integrative study of the expression and mutational status of genes related to iron and mitochondrial metabolism was carried out. A total of 231 low-risk MDS patients and 81 controls were studied. Gene expression analysis revealed that iron metabolism and mitochondrial function had the highest number of genes deregulated in RARS patients compared to controls and the refractory cytopenias with unilineage dysplasia (RCUD). Thus mitochondrial transporters SLC25 (SLC25A37 and SLC25A38) and ALAD genes were over-expressed in RARS. Moreover, significant differences were observed between patients with SF3B1 mutations and patients without the mutations. The deregulation of genes involved in iron and mitochondrial metabolism provides new insights in our knowledge of MDS-RS. New variants that could be involved in the pathogenesis of these diseases have been identified.

Urothelial cancer gene regulatory networks inferred from large-scale RNAseq, Bead and Oligo gene expression data

BACKGROUND: Urothelial pathogenesis is a complex process driven by an underlying network of interconnected genes. The identification of novel genomic target regions and gene targets that drive urothelial carcinogenesis is crucial in order to improve our current limited understanding of urothelial cancer (UC) on the molecular level. The inference of genome-wide gene regulatory networks (GRN) from large-scale gene expression data provides a promising approach for a detailed investigation of the underlying network structure associated to urothelial carcinogenesis.

METHODS: In our study we inferred and compared three GRNs by the application of the BC3Net inference algorithm to large-scale transitional cell carcinoma gene expression data sets from Illumina RNAseq (179 samples), Illumina Bead arrays (165 samples) and Affymetrix Oligo microarrays (188 samples). We investigated the structural and functional properties of GRNs for the identification of molecular targets associated to urothelial cancer.

RESULTS: We found that the urothelial cancer (UC) GRNs show a significant enrichment of subnetworks that are associated with known cancer hallmarks including cell cycle, immune response, signaling, differentiation and translation. Interestingly, the most prominent subnetworks of co-located genes were found on chromosome regions 5q31.3 (RNAseq), 8q24.3 (Oligo) and 1q23.3 (Bead), which all represent known genomic regions frequently deregulated or aberated in urothelial cancer and other cancer types. Furthermore, the identified hub genes of the individual GRNs, e.g., HID1/DMC1 (tumor development), RNF17/TDRD4 (cancer antigen) and CYP4A11 (angiogenesis/ metastasis) are known cancer associated markers. The GRNs were highly dataset specific on the interaction level between individual genes, but showed large similarities on the biological function level represented by subnetworks. Remarkably, the RNAseq UC GRN showed twice the proportion of significant functional subnetworks. Based on our analysis of inferential and experimental networks the Bead UC GRN showed the lowest performance compared to the RNAseq and Oligo UC GRNs.

CONCLUSION: To our knowledge, this is the first study investigating genome-scale UC GRNs. RNAseq based gene expression data is the data platform of choice for a GRN inference. Our study offers new avenues for the identification of novel putative diagnostic targets for subsequent studies in bladder tumors.

The impact of next generation sequencing technologies on haematological research - A review

Next-generation sequencing (NGS) technologies have begun to revolutionize the field of haematological malignancies through the assessment of a patient's genetic makeup with a minimal cost. Significant discoveries have already provided a unique insight into disease initiation, risk stratification and therapeutic intervention. Sequencing analysis will likely form part of the routine diagnostic testing in the future. However, a number of important issues need to be addressed for that to become a reality with regard to result interpretation, laboratory workflow, data storage and ethical issues. In this review we summarize the contribution that NGS has already made to the field of haematological malignancies. Finally, we discuss the challenges that NGS technologies will bring in relation to data storage, ethical and legal issues and laboratory validation. Despite these challenges, we predict that high-throughput DNA sequencing will redefine haematological malignancies based on individualized genomic analysis.

Primary airway epithelial cell culture and asthma in children-lessons learnt and yet to come

Until recently the airway epithelial cell (AEC) was considered a simple barrier that prevented entry of inhaled matter into the lung parenchyma. The AEC is now recognized as having an important role in the inflammatory response of the respiratory system to inhaled exposures, and abnormalities of these responses are thought to be important to asthma pathogenesis. This review first explores how the challenges of studying nasal and bronchial AECs in children have been addressed and then summarizes the results of studies of primary AEC function in children with and without asthma. There is good evidence that nasal AECs may be a suitable surrogate for the study of certain aspects of bronchial AEC function, although bronchial AECs remain the gold standard for asthma research. There are consistent differences between children with and without asthma for nasal and bronchial AEC mediator release following exposure to a range of pro-inflammatory stimulants including interleukins (IL)-1β, IL-4, and IL-13. However, there are inconsistencies between studies, e.g., release of IL-6, an important pro-inflammatory cytokine, is not increased in children with asthma relative to controls in all studies. Future work should expand current understanding of the "upstream" signalling pathways in AEC, study AEC from children before the onset of asthma symptoms and in vitro models should be developed that replicate the in vivo status more completely, e.g., co-culture with dendritic cells. AECs are difficult to obtain from children and collaboration between centers is expected to yield meaningful advances in asthma understanding and ultimately help deliver novel therapies.