Development of new molecular methods for the diagnosis and the study of viral diseases of fish

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.

Neurodegenerative diseases: molecular mechanisms and new strategies for neuroprotection

The term neurodegeneration defines numerous conditions that modify neuron’s normal functions in the human brain where is possible to observe a progressive and consistent neuronal loss. The mechanisms involved in neurodegenerative chronic and acute diseases evolution are not completely understood yet, however they share common characteristics such as misfolded proteins, oxidative stress, inflammation, excitotoxicity, and neuronal loss. Many studies have shown the frequency to develop neurodegenerative chronic diseases several years after an acute brain injury. In addition, many patients show, after a traumatic brain injury, motor and cognitive manifestations that are close to which are observed in neurodegenerative chronic patients. For this reason it is evident how is fundamental the concept of neuroprotection as a way to modulate the neurodegenerative processes evolution. Neuroinflammation, oxidative stress and the apoptotic process may be functional targets where operate to this end. Taking into account these considerations, the aim of the present study is to identify potential common pathogenetic pathways in neurodegenerative diseases using an integrated approach of preclinical studies. The goal is to delineate therapeutic strategies for the prevention of neuroinflammation, neurodegeneration and dysfunctions associated to Parkinson’s disease (PD) and cerebral ischemia. In the present study we used a murine model of PD treated with an isothiocyanate, 6-MSITC, able to quench ROS formation, restore the antioxidant GSH system, slow down the apoptotic neuronal death and counteract motor dysfunction induced by 6-OHDA. In the second study we utilized a transgenic mouse model knockout for CD36 receptor to investigate the inflammation involvement in a long term study of MCAo, which shows a better outcome after the damage induced. In conclusion, results in this study allow underlying the connection among these pathologies, and the importance of a neuroprotective strategy able to restore neurons activity where current drugs therapies have shown palliative but not healing abilities.

Blood-CNS barriers in neurodegenerative diseases

The blood-brain barrier (BBB) and the blood-spinal cord barrier (BSCB) separate the brain and the spinal cord from the circulating blood and are important for the maintenance of the CNS homeostasis. They build a physical barrier thereby protecting the CNS from pathogens and toxic agents, and their disruption plays a crucial role in the pathogenesis of several CNS disorders. In this thesis, the blood-CNS-barriers were studied via in vitro models in two case studies for neurodegenerative disorders, in particular Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). The first model evaluates treatment possibilities of AD using nanotechnology-based strategies. Since the toxic amyloid-β42 (Aβ42) peptide plays a crucial role in the pathogenesis of AD, reduced generation or enhanced clearance of Aβ42 peptides are expected to modify the disease course in AD. Therefore, several Aβ42-lowering drugs like flurbiprofen had been tested in clinical trials, but most of them failed due to their low brain penetration. Here, flurbiprofen was embedded in polylactide (PLA) nanoparticles and its transport was examined in an in vitro BBB model. The embedding of flurbiprofen into the nanoparticles disguised its cytotoxic potential and enabled the administration of higher drug concentrations which resulted in a sufficient transport of the drug across an endothelial cell monolayer. These results demonstrate that non-permeable drugs can be transported efficiently via nanoparticles and that these nanotechnology-based strategies are a promising tool to generate novel therapeutic options for AD and other CNS diseases. rnThe focus of the second project was to investigate the impaired integrity of the BSCB in a mouse model for ALS. About 20% of all familial ALS cases are associated with missense mutations or small deletions in the gene that encodes Cu/Zn-superoxide dismutase 1 (SOD1). To date, the molecular mechanisms resulting in ALS are still unknown, but there is evidence that the disruption of the BSCB is one of the primary pathological events. In both familial and sporadic ALS patients, loss of endothelial integrity and endothelial cell damage was observed, and studies with SOD1 transgenic mice demonstrated that the BSCB disruption was found prior to motor neuron degeneration and neurovascular inflammation. Thus, an in vitro model for ALS endothelial cells was generated which exhibited comparable integrity characteristics and tight junction (TJ) protein expression profiles as isolated primary endothelial cells of the BSCB of SOD1 transgenic mice. In this, an alteration of the βcat/AKT/FoxO1 pathway, which regulates the expression of the TJ protein claudin-5, could be observed. These data furthermore indicate that ALS is a neurovascular disease, and understanding of the primary events in ALS pathogenesis will hopefully provide ideas for the development of new therapeutic strategies. rn

In vitro detection of cytotoxic T and NK cells in peripheral blood of patients with various drug-induced skin diseases

BACKGROUND: Cytotoxic cells are involved in most forms of drug-induced skin diseases. Till now, no in vitro test addressed this aspect of drug-allergic responses. Our report evaluates whether drug-induced cytotoxic cells can be detected in peripheral blood of nonacute patients with different forms of drug hypersensitivity, and also whether in vitro detection of these cells could be helpful in drug-allergy diagnosis. METHODS: GranzymeB enzyme-linked immunosorbent spot-forming (ELISPOT) and cell surface expression of the degranulation marker CD107a were evaluated on peripheral blood mononuclear cells from 12 drug-allergic patients in remission state and 16 drug-exposed healthy controls. RESULTS: In 10/12 allergic patients culprit but not irrelevant drug elicited granzymeB release after 48-72 h stimulation. It was clearly positive in patients with high proliferative response to the drug, measured in lymphocyte transformation tests. In patients, who showed moderate or low proliferation and low drug-response in granzymeB ELISPOT, overnight preincubation with interleukin (IL)-7/IL-15 enhanced drug-specific granzymeB release and allowed to clearly identify the offending agent. CD107a staining was positive on CD4+/CD3+, CD8+/CD3+ T cells as well as CD56+/CD3- natural killer cells. None of the drug-exposed healthy donors reacted to the tested drugs and allergic patients reacted only to the offending, but not to tolerated drugs. CONCLUSION: GranzymeB ELISPOT is a highly specific in vitro method to detect drug-reacting cytotoxic cells in peripheral blood of drug-allergic patients even several years after disease manifestation. Together with IL-7/IL-15 preincubation, it may be helpful in indentifying the offending drug even in some patients with weak proliferative drug-response.

[Swiss registry for TNF-alpha blockers in children and adolescents with rheumatological diseases]

We created a registry to evaluate long term outcome, efficacy and adverse events for children treated wit TNF-alpha inhibitors in Switzerland. 106 patients (68 female/38 male) were included. 61 patients were treated with Etanercept (Enbrel) and 45 with Infliximab (Remicade). Concomitant treatment at baseline included corticosteroids in 26% and Methotrexate in 75% of the patients. Subjective disease activity three months after initiation of TNF-alpha was better in 81%, worse in 4% and stable in 15% of the patients. In total 24 adverse events in 21 patients were reported. Treatment with TNF-alpha inhibitors seems to be safe and effective for children and adolescents with rheumatologic diseases.

Expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases

BACKGROUND: Inflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases. METHODS: sCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. In vitro studies were performed to investigate which factors regulate sCD14 release and mCD14 expression. RESULTS: sCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. In vitro, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition. CONCLUSIONS: This study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.

Spectral-domain optical coherence tomography use in macular diseases: a review

The introduction of spectral-domain optical coherence tomography (SD-OCT) has improved the clinical value for assessment of the eyes with macular disease. This article reviews the advances of SD-OCT for the diagnostic of various macular diseases. These include vitreomacular traction syndrome, cystoid macular edema/diabetic macular edema, epiretinal membranes, full-thickness macular holes, lamellar holes, pseudoholes, microholes, and schisis from myopia. Besides offering new insights into the pathogenesis of macular abnormalities, SD-OCT is a valuable tool for monitoring macular disease.

[Molecular analyses for (early) recognition of hematologic diseases - sense and sensibility for molecular analysis: the art of intelligent decision-making]

During the last 10 years several molecular markers have been established as useful tools among the armamentarium of a hematologist. As a consequence, the number of performed hematologic molecular analyses has immensely increased. Often, such tests replace or complement other laboratory methods. Molecular markers can be useful in many ways: they can serve for diagnostics, describe the prognostic profile, predict which types of drugs are indicated, and can be used for the therapeutic monitoring of the patient to indicate an adequate response or predict resistance or relapse of the disease. Many markers fulfill more than one of these aspects. Most important, however, is the right choice of analyses at the right time-points!

Pregnancy and reproduction in autoimmune rheumatic diseases

Despite evidence for the important role of oestrogens in the aetiology and pathophysiology of chronic immune/inflammatory diseases, the previous view of an unequivocal beneficial effect of oestrogens on RA compared with a detrimental effect on SLE has to be reconsidered. Likewise, the long-held belief that RA remits in the majority of pregnant patients has been challenged, and shows that only half of the patients experience significant improvement when objective disease activity measurements are applied. Pregnancies in patients with SLE are mostly successful when well planned and monitored interdisciplinarily, whereas a small proportion of women with APS still have adverse pregnancy outcomes in spite of the standard treatment. New prospective studies indicate better outcomes for pregnancies in women with rare diseases such as SSc and vasculitis. Fertility problems are not uncommon in patients with rheumatic disease and need to be considered in both genders. Necessary therapy, shortly before or during the pregnancy, demands taking into account the health of both mother and fetus. Long-term effects of drugs on offspring exposed in utero or during lactation is a new area under study as well as late effects of maternal rheumatic disease on children.

Measurement issues in trials of pediatric acute diarrheal diseases: a systematic review

BACKGROUND: Worldwide, diarrheal diseases rank second among conditions that afflict children. Despite the disease burden, there is limited consensus on how to define and measure pediatric acute diarrhea in trials. OBJECTIVES: In RCTs of children involving acute diarrhea as the primary outcome, we documented (1) how acute diarrhea and its resolution were defined, (2) all primary outcomes, (3) the psychometric properties of instruments used to measure acute diarrhea and (4) the methodologic quality of included trials, as reported. METHODS: We searched CENTRAL, Embase, Global Health, and Medline from inception to February 2009. English-language RCTs of children younger than 19 years that measured acute diarrhea as a primary outcome were chosen. RESULTS: We identified 138 RCTs reporting on 1 or more primary outcomes related to pediatric acute diarrhea/diseases. Included trials used 64 unique definitions of diarrhea, 69 unique definitions of diarrhea resolution, and 46 unique primary outcomes. The majority of included trials evaluated short-term clinical disease activity (incidence and duration of diarrhea), laboratory outcomes, or a composite of these end points. Thirty-two trials used instruments (eg, single and multidomain scoring systems) to support assessment of disease activity. Of these, 3 trials stated that their instrument was valid; however, none of the trials (or their citations) reported evidence of this validity. The overall methodologic quality of included trials was good. CONCLUSIONS: Even in what would be considered methodologically sound clinical trials, definitions of diarrhea, primary outcomes, and instruments employed in RCTs of pediatric acute diarrhea are heterogeneous, lack evidence of validity, and focus on indices that may not be important to participants.

Stem cell-based therapeutic applications in retinal degenerative diseases

Retinal degenerative diseases that target photoreceptors or the adjacent retinal pigment epithelium (RPE) affect millions of people worldwide. Retinal degeneration (RD) is found in many different forms of retinal diseases including retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy, cataracts, and glaucoma. Effective treatment for retinal degeneration has been widely investigated. Gene-replacement therapy has been shown to improve visual function in inherited retinal disease. However, this treatment was less effective with advanced disease. Stem cell-based therapy is being pursued as a potential alternative approach in the treatment of retinal degenerative diseases. In this review, we will focus on stem cell-based therapies in the pipeline and summarize progress in treatment of retinal degenerative disease.

Adrenal insufficiency after glucocorticoid withdrawal in children with rheumatic diseases

?Glucocorticoids (GCs) are often used for the treatment of rheumatic disorders. However, doses are prescribed, which may suppress the hypothalamic-pituitary-adrenal (HPA) axis. After GC withdrawal, recovery of the HPA axis may be delayed putting the patient at risk for adrenal insufficiency. We assessed adrenal function and factors influencing adrenal responsiveness after termination of GC therapy in paediatric patients with rheumatic diseases.