Rift Valley fever virus detection in susceptible hosts with special emphasis in insects.

Rift Valley fever phlebovirus (RVFV, Phenuiviridae) is an emerging arbovirus that can cause potentially fatal disease in many host species including ruminants and humans. Thus, tools to detect this pathogen within tissue samples from routine diagnostic investigations or for research purposes are of major interest. This study compares the immunohistological usefulness of several mono- and polyclonal antibodies against RVFV epitopes in tissue samples derived from natural hosts of epidemiologic importance (sheep), potentially virus transmitting insect species (Culex quinquefasciatus, Aedes aegypti) as well as scientific infection models (mouse, Drosophila melanogaster, C6/36 cell pellet). While the nucleoprotein was the epitope most prominently detected in mammal and mosquito tissue samples, fruit fly tissues showed expression of glycoproteins only. Antibodies against non-structural proteins exhibited single cell reactions in salivary glands of mosquitoes and the C6/36 cell pellet. However, as single antibodies exhibited a cross reactivity of varying degree in non-infected specimens, a careful interpretation of positive reactions and consideration of adequate controls remains of critical importance. The results suggest that primary antibodies directed against viral nucleoproteins and glycoproteins can facilitate RVFV detection in mammals and insects, respectively, and therefore will allow RVFV detection for diagnostic and research purposes.

Differential effects of basolateral and apical iron supply on iron transport in Caco-2 cells.

Iron homeostasis in the human body is maintained primarily through regulation of iron absorption in the duodenum. The liver peptide hepcidin plays a central role in this regulation. Additionally, expression and functional control of certain components of the cellular iron transport machinery can be influenced directly by the iron status of enterocytes. The significance of this modulation, relative to the effects of hepcidin, and the comparative effects of iron obtained directly from the diet and/or via the bloodstream are not clear. The studies described here were performed using Caco-2 cell monolayers as a model of intestinal epithelium, to compare the effects of iron supplied in physiologically relevant forms to either the apical or basolateral surfaces of the cells. Both sources of iron provoked increased cellular ferritin content, indicating iron uptake from both sides of the cells. Supply of basolateral transferrin-bound iron did not affect subsequent iron transport across the apical surface, but reduced iron transport across the basolateral membrane. In contrast, the apical iron supply led to subsequent reduction in iron transport across the apical cell membrane without altering iron export across the basolateral membrane. The apical and basolateral iron supplies also elicited distinct effects on the expression and subcellular distribution of iron transporters. These data suggest that, in addition to the effects of cellular iron status on the expression of iron transporter genes, different modes and direction of iron supply to enterocytes can elicit distinct functional effects on iron transport.

Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism.

There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.

Quadrimembral amputation: indications and contraindications for vascularized composite allotransplantation.

INTRODUCTION: Quadrimembral amputees, as patients who have lost both upper and lower extremities, may benefit greatly from hand transplantation. The objective of this study is to evaluate the indications and contraindications for transplantation in this subset of patients. METHODS: A retrospective review was conducted of five quadrimembral amputees evaluated by our program for transplantation. Information collected included age, sex, level of amputations, time since amputations, etiology, level of dependence, medical stability, psychosocial status, and the ability to tolerate immunosuppression. Indications and contraindications for transplantation were reviewed for each patient. RESULTS: All etiologies were based in extremity ischemia: three from septic shock, one from myocardial infarction, and one from drug overdose. All patients are completely dependent. Of the five patients, two needed further reconstructive surgery and two others had a history of resolved hepatic/renal insufficiency. After thorough evaluation, two patients were selected as potential transplant candidates. They demonstrated strong psychosocial support systems, a thorough understanding of hand transplantation, along with its risks and postoperative requirements. They had also completed a full regimen of rehabilitation along with prosthetic fitting and utilization. CONCLUSIONS: Clearance for transplantation is based on medical stability, absence of infection or systemic diseases, and strong psychosocial support systems. Contraindications for transplantation are drug dependence and noncompliant behavior. Relative contraindications include a history of hepatic/renal insufficiency which if not resolved may preclude the use of postoperative immunosuppression.

The impact of clinical factors on the development of late radiation toxicity: results from the Medical Research Council RT01 trial (ISRCTN47772397).

AIMS: A variety of dosimetric parameters have been shown to influence the incidence of late radiation toxicity. The effect of other treatment- and patient-related factors is less well established. The aim of this study was to elucidate the influence of such factors in the development of late symptoms after radical radiotherapy to the prostate. MATERIALS AND METHODS: Patient- and treatment-related factors that are thought to influence the development of late toxicity were analysed in 788 patients who had received radical radiotherapy to the prostate in the Medical Research Council RT01 trial. Late toxicity data were recorded using the Radiation Therapy Oncology Group, Late Effects of Normal Tissues/Subjective, Objective, Management, Analytic, Royal Marsden Hospital and the University of California, Los Angeles, Prostate Cancer Index. Acute toxicity was measured using the Radiation Therapy Oncology Group grading system. RESULTS: On multivariate analysis, acute bowel toxicity was statistically significantly associated with increased proctitis (hazard ratio=1.63, 95% confidence interval 1.18, 2.24; P=0.003) and increased stool frequency (hazard ratio=1.77, 95% confidence interval 1.27, 2.46; P=0.001). Hypertension was strongly associated with a decreased risk of poor urinary stream (hazard ratio=0.25, 95% confidence interval 0.09, 0.71; P=0.009). There was an increased risk of rectal bleeding with increased age (hazard ratio=1.04 per year of age, 95% confidence interval 1.01, 1.08; P=0.009). As expected, a higher prescribed dose increased the risk of several late toxicity end points. Although acute bladder toxicity was associated with the presence of bladder symptoms at 5 years, the effect disappeared for all symptoms except increased urinary frequency and haematuria when a change in bladder function from baseline was calculated. Patients with any pretreatment bladder symptoms were more likely to report increased urinary frequency (hazard ratio=2.09, 95% confidence interval 1.48, 2.95; P<0.0005), increased urinary incontinence (hazard ratio=4.22, 95% confidence interval 2.13, 8.35; P<0.0005) and decreased stream (hazard ratio=2.64, 95% confidence interval 1.62, 4.31; P<0.0005), after treatment and before the most recent follow-up assessment. CONCLUSIONS: In this study, increased acute gastrointestinal and bladder symptoms and prescribed dose were associated with increased late radiation toxicity. The presence of hypertension seemed to be protective for the development of late effects. Baseline symptoms should be taken into account when radiation toxicity is analysed.

Bunyaviruses and climate change.

It is generally accepted that the planet is undergoing climatic changes, and 'climate change' has become the scapegoat for many catastrophes, including infectious disease outbreaks, as acknowledged by Randolph and Ergonul, who state 'Climate change is the current ubiquitous explanation for increased incidence of infections of many sorts' (Future Virology 2008; 3: 303-306). However, as these authors argue, this is a highly simplistic view and, indeed, there is a complex network of factors that are responsible for disease emergence and re-emergence. In this short review, the role that climate change could play in the emergence of bunyavirus disease is considered, using a few selected examples.

Effects of oviductal proteins, including heat shock 70 kDa protein 8, on survival of ram spermatozoa over 48 h in vitro.

Following insemination, ram spermatozoa are transported to the isthmus region of the oviduct where they bind to the oviductal epithelial cells (OEC), remaining viable for several hours. The aim of the present study was to begin to decipher which component(s) of the ewe oviduct actively participates in maintaining the viability of ram spermatozoa. A series of experiments was conducted to investigate whether: (1) soluble OEC apical plasma membrane proteins (sAPM) isolated from ewes prolong survival of ram spermatozoa over an extended (48 h) coincubation period at 39 degrees C; (2) a recombinant form of one of these oviductal proteins, namely heat shock 70 kDa protein 8 (HSPA8), prolongs survival of ram spermatozoa; and (3) pretreatment with HSPA8 antibody compromises the ability of sAPM to prolong the survival of ram spermatozoa. Both sAPM and recombinant HSPA8 had a beneficial effect on the viability of ram spermatozoa during coincubation, although both these effects were dose dependent. In contrast, pretreatment with HSPA8 antibody significantly negated the ability of sAPM to maintain the viability of ram spermatozoa. These findings suggest that HSPA8 is an active component of the ewe oviduct that participates in maintaining the viability of ram spermatozoa. This is a potentially valuable observation given that there is a great deal of room for improving existing diluents for storing fresh ram semen.

Role of the NSs protein in the zoonotic capacity of Orthobunyaviruses.

The family Bunyaviridae contains over 350 named isolates, classified into five genera: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. The Orthobunyavirus genus contains some 170 isolates that are mainly transmitted by mosquitoes and are responsible for a range of disease syndromes in humans including self-limiting febrile illness, encephalitis and haemorrhagic fever. The viruses have a tripartite, negative-sense RNA genome. Analyses of viruses in four serogroups (Bunyamwera, California, Group C and Simbu) showed that the smallest (S) RNA segment encodes the nucleocapsid protein (N) and a non-structural protein called (NSs). The NSs protein of Bunyamwera virus (BUNV) has been shown to play a role in shut-off of host cell protein synthesis in mammalian cells, but no protein shut-off is observed in BUNVinfected mosquito cells (Aedes albopictus C6/36 cells). Protein shut-off in infected mammalian cells is achieved by global inhibition of RNA polymerase II-mediated transcription and enables the virus to overcome the host innate immune response. As innate defence mechanisms constitute a significant barrier to virus infection of different hosts, NSs would appear to play a key role in determining the zoonotic capacity of orthobunyaviruses.

Oligonucleotide microarray analysis of human lens epithelial cells: TGFbeta regulated gene expression.

PURPOSE: Transforming growth factor beta (TGFbeta), a pro-fibrotic cytokine has been proposed a causative factor in the progression of lens pathologies including posterior capsule opacification (PCO), a condition that occurs after cataract surgery. This study employs oligonucleotide microarrays to provide a global profile of gene expression in FHL 124 cells, to identify changes in gene expression following treatment with TGFbeta1 and TGFbeta2, and to enable putative genes relating to TGFbeta regulation and PCO to be identified. METHODS: Routinely cultured FHL 124 cells maintained in serum free Eagle's Minimum Essential Medium (EMEM) were treated with either TGFbeta1 or TGFbeta2 at 10 ng/ml for 24 h then total RNA extraction was carried out. Total RNA (16 microg) was used to analyze gene expression by spotted oligonucleotide microarray hybridization. The spotted oligonucleotide microarrays employed contained 13,971 oligonucleotide probes, each designed to be specific for an individual gene. Array images were analyzed using GenePix Pro 3.0, followed by raw data import into GeneSpring 7.0 where a cross gene error model (CGEM) filter was applied. Data was subjected to LoWess normalization prior to comparison of the different treatment groups. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to validate the oligonucleotide microarray data, using a select number of genes exhibiting differential expression. RESULTS: A total of 301 genes were up-regulated by more than 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these up-regulated genes had biological functions relevant to lens epithelial cells including roles in contraction, transdifferentiation and as extracellular matrix (ECM) components. A total of 164 genes were down-regulated by more that 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these down-regulated genes have biological functions including roles in apoptosis, signaling, and as anti-oxidants. Following treatment with TGFbeta1 and TGFbeta2, QRT-PCR successfully validated the differential changes in gene expression detected by oligonucleotide microarrays. CONCLUSIONS: TGFbeta1 and TGFbeta2 regulate the gene expression of genes that have important roles in human lens epithelial cell biology. Most importantly, TGFbeta induces the gene expression of a number of fibrotic markers which may have a role in promoting the development of PCO such as transdifferentiation markers, contractile factors, and ECM components.

The genomics revolution and radiotherapy.

The expansion of our knowledge through the Human Genome Project has been accompanied by the development of new high-throughput techniques, which provide extensive capabilities for the analysis of a large number of genes or the whole genome. These assays can be carried out in various clinical samples at the DNA (genome), RNA (transcriptome) or protein (proteome) level. There is a belief that this genomic revolution, i.e. sequencing of the human genome and developments in high-throughput technology, heralds a future of personalised medicine. For clinical oncology, this progress should increase the possibility of predicting individual patient responses to radiotherapy. This review highlights some of the work involving sparsely ionising radiation and the new technologies.

Translation efficiencies of the 5'-untranslated region of genotypes 1a and 3a in hepatitis C infected patients.

Differences between the translation efficiencies mediated by the 5'-untranslated regions (5'-UTR) of genotypes (gt) 1 and 3 of hepatitis C virus (HCV) have been reported but it is unknown if such differences are biologically significant. The 5'-UTR was sequenced from paired serum and liver samples from 26 patients with chronic HCV hepatitis (11 gt 1a, 15 gt 3a). To determine whether there is a consistent difference between gts 1a and 3a translation efficiency, 5'-UTR (nt 1-356) and 5'-UTR plus core (nt 1-914) sequences were cloned into bicistronic, luciferase-encoding constructs and relative translation efficiencies (RTE) measured in Huh7 cells and BHK cells. The relationships between viral load, liver biopsy Ishak scores, degree of steatosis and translational activity of the patient-derived nucleotide sequence were examined. There were no differences in 5'-UTR sequence between serum and corresponding liver samples. The mean RTE of 5'-UTR sequences from gt 3a isolates was not significantly different from gt 1a whether or not the core encoding sequence was included, although inclusion of core led to a reduction in RTE by 93-97% for both genotypes. No correlation was found between RTE and serum HCV RNA levels, liver steatosis, inflammation, or fibrosis. However, a significant correlation was found between the presence of steatosis and infection with HCV gt 3a. It is concluded that there was no difference in translation efficiencies of 5'-UTRs from patients infected with gts 1a and 3a, and translation activity measured in vitro does not correlate with viral load or severity of liver disease.

Harnessing the biology of the oviduct for the benefit of artificial insemination.

Spermatozoa fulfil a single role, namely achieving syngamy by transporting the haploid genome to their counterpart gamete, the oocyte. Simple as this may seem, it is fraught with many difficulties, especially in the face of biological processes that enable females to select spermatozoa after they have mated multiply with several males. Conversely, the female reproductive tract sequesters a privileged sperm subpopulation in the oviductal isthmus for variable periods of time, releasing them when the time is opportune for fertilisation. Recent studies of sperm transport in the female reproductive tract suggest that these phenomena involve signalling dialogues between spermatozoa and the female reproductive tract environment. Opportunities for mutual signalling are immense but have received relatively little attention. The oviduct is an organ of crucial significance in modulating sperm function and may be one of the most important sites for determining many aspects of sperm selection and competition. The oviductal environment possesses the potential for enhancing sperm survival, suppressing and activating sperm motility as required, and responds to the arrival of spermatozoa by producing novel proteins. While the biological nature of the sperm-oviduct dialogue is interesting for its own sake, the mechanisms that govern these processes offer opportunities for the improvement of artificial insemination procedures. If oviductal proteins enhance sperm survival, they offer opportunities for the development of long-life semen diluents. Conversely, if we understood the basis of sperm selection we may be able to concentrate on identifying and using only the best sperm subpopulations for improved animal breeding efficiency.

Correlation between translation efficiency and outcome of combination therapy in chronic hepatitis C genotype 3.

Combination therapy with interferon-alpha (IFN-alpha) and ribavirin (RBV) in chronic hepatitis C demonstrates the best responses against hepatitis C virus (HCV) of genotype 3. Still, it has proven to be ineffective in 20-30% of patients infected with this genotype. In the present study, we analysed the translation efficiency mediated by the internal ribosome entry site (IRES) region in HCV genotype 3 genomes isolated from sustained responders (SR) and non-responders (NR), assuming that this may influence the outcome of treatment. Pretreatment isolates of genotype 3 from 22 individuals (15 SR, seven NR) were selected for such analyses. The IRES region [nucleotide (nt) 1-407] was cloned into a dual luciferase vector and IRES activity assessed following transfection into various cell lines. Low relative translation efficiency was observed for IRES elements derived from SR patients, whereas those of NR patients showed significantly greater translation efficiency (29.7 +/- 13 vs 69.4 +/- 22; P < 0.01). Subsequently, the effect of IFN-alpha plus RBV on IRES-driven translation in vitro was determined. A greater suppressive effect was observed on IRES activity isolated from seven SR patients, when compared with seven NR patients. In conclusion, IRES efficiency in vitro correlated with treatment response for HCV genotype 3. Further studies are warranted to investigate whether IRES efficiency in vitro, or sequence motifs associated with IRES efficiency, will be worthwhile to explore as prognostic tools for other HCV genotypes in the treatment of chronic HCV infection.

Validation of an experimental strategy for studying surface-exposed proteins involved in porcine sperm-oviduct contact interactions.

Previous experiments have shown that boar sperm survival in vitro is enhanced when co-incubated with a solubilised protein extract of porcine oviducal apical plasma membrane proteins. Here, we examine the hypothesis that the effects are mediated by direct oviduct-sperm contact and use in situ biotinylation of the oviducal epithelial surface to trace the surface-exposed biotinylated proteins through purification and solubilisation steps. We have also examined the effectiveness of mechanical scraping as a method of recovering oviducal epithelial proteins. We show that a subset of proteins originally exposed at the oviducal surface eventually bind to spermatozoa during incubation in vitro, but also show that a different protein subset is implicated if the sperm incubation is performed with proteins that had been biotinylated after (ex situ) extraction from the oviduct. Apical plasma membrane fractions biotinylated after purification contained many more biotinylated protein bands than preparations labelled before purification and multiple protein bands were eventually found to associate with spermatozoa. Although the evidence presented here supports the hypothesis that protein(s) anchored to the oviducal epithelium bind populations of spermatozoa directly and may have a role in the enhancement of sperm viability, it also shows that the choice of investigative technique exerts a major influence on experimental outcomes.

In vitro maintenance of boar sperm viability by a soluble fraction obtained from oviductal apical plasma membrane preparations.

Oviductal apical plasma membrane fractions have been successfully used to provide an in vitro model to study the role of direct membrane contact in sperm-oviduct interactions. Apical plasma membrane preparations from pig oviductal tissues show a dose-response in their ability to maintain boar sperm viability in vitro. Membrane preparations obtained from other tissues (lung and duodenum) are incapable of maintaining boar sperm viability to the same extent as oviductal tissue. The present study examined the validity of two hypotheses that arise from current knowledge of sperm-oviduct interactions, namely, that (i) apical plasma membranes prepared from ampullar regions of the oviduct are less effective than those from isthmus regions, and (ii) sperm survival is more effective in apical plasma membrane preparations derived from follicular phase oviducts than those derived from luteal phase oviducts. Both hypotheses were proved false. The nature of the active component(s) in the oviductal apical plasma membrane fractions was further investigated. Heat treatment (100 degrees C for 20 min) diminished the capacity of membranes to support boar sperm viability. Furthermore, a soluble salt-extracted fraction obtained from oviductal apical plasma membrane preparations was biologically active and supported boar sperm viability in vitro. This may indicate that the active factor(s) responsible for the maintenance of boar sperm viability is not an integral part of oviductal membranes and is peripherally bound to these membranes.