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.

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.

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.

Calcium-binding proteins from the outer acrosomal membrane of ram spermatozoa: potential candidates for involvement in the acrosome reaction.

After an intracellular calcium influx, fusion of the sperm plasma membrane and outer acrosomal membrane (the acrosome reaction) precedes mammalian fertilization in vivo. This study describes the isolation of outer acrosomal membrane from ram spermatozoa and the subsequent characterization of calcium-binding proteins. Pooled ejaculates were diluted, cooled slowly and washed. Incubation with Hyamine 1622 (benzethenium chloride) and subsequent slow centrifugation gently dislodged and concentrated acrosomal membranes, the fragments of which were isolated on a two-step discontinuous sucrose gradient. The acrosomal membrane material stained with Giemsa, whereas spermatozoa from the gradient pellet stained intensely only in the equatorial segment. The acrosomal fraction showed a limited number of polypeptides by SDS-PAGE. Incubation with 45Ca2+ revealed two radioactive bands at 34 and 39 kDa. Extraction in the presence of EGTA implied that these proteins are not peripheral proteins associated with the membrane only in the presence of calcium ions, but are integral membrane proteins. Polyclonal antisera raised to the two bands showed specific binding to the anterior acrosomal region and demonstrated the intracellular location of the proteins. Sequence data of protein A revealed 83% homology with calnexin homologue precursor and 70% homology with annexin XI. Protein B showed 68% homology with protein SP-10 precursor and 64-72% homology with various annexins. However, crossreactivity with a range of commercial annexin antibodies and a specific antibody to a synthetic motif encompassing the annexin calcium-binding site was not demonstrable. It is concluded that the isolated proteins are unlikely to be annexins, but are possibly novel calcium-binding proteins.

Measurement of cellular repair activities for oxidative DNA damage.

Two related assays capable of determining cell extract repair activities for different oxidative lesions in DNA are described. Both assays measure the incorporation of radiolabeled nucleotides during repair of an oxidatively damaged template in a cell-free system. The assays differ in the type of oxidative damage present in the DNA. In one, singlet oxygen is used to generate predominantly 8-oxo-2'-deoxyguanosine lesions. In the other, hydroxyl radicals are used to generate a broad spectrum of damage including oxidized bases and strand breaks. Assay conditions were adjusted to ensure that radiolabel incorporation was directly proportional to cell extract repair activity. These assays represent sensitive tools for investigating the regulation of repair systems for oxidative DNA damage.

MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability.

DNA mismatch repair is important because of its role in maintaining genomic integrity and its association with hereditary non-polyposis colon cancer (HNPCC). To identify new human mismatch repair proteins, we probed nuclear extracts with the conserved carboxy-terminal MLH1 interaction domain. Here we describe the cloning and complete genomic sequence of MLH3, which encodes a new DNA mismatch repair protein that interacts with MLH1. MLH3 is more similar to mismatch repair proteins from yeast, plants, worms and bacteria than to any known mammalian protein, suggesting that its conserved sequence may confer unique functions in mice and humans. Cells in culture stably expressing a dominant-negative MLH3 protein exhibit microsatellite instability. Mlh3 is highly expressed in gastrointestinal epithelium and physically maps to the mouse complex trait locus colon cancer susceptibility I (Ccs1). Although we were unable to identify a mutation in the protein-coding region of Mlh3 in the susceptible mouse strain, colon tumours from congenic Ccs1 mice exhibit microsatellite instability. Functional redundancy among Mlh3, Pms1 and Pms2 may explain why neither Pms1 nor Pms2 mutant mice develop colon cancer, and why PMS1 and PMS2 mutations are only rarely found in HNPCC families.

Oxidative insult specifically decreases levels of a mitochondrial transcript.

The effects of oxidative insult, applied with hydrogen peroxide, on gene transcript levels in a human lymphocyte cell line (Molt-17) were investigated using mRNA differential display. Several cDNA fragments corresponding to putatively up- or down-regulated transcripts were isolated. One of these was found to hybridize to two discrete transcripts on Northern blots of Molt-17 cell RNA. The more abundant transcript, that has previously been demonstrated to correspond to the mRNA for mitochondrial ATPase subunits 8 and 6, was unaffected by the hydrogen peroxide treatment. In contrast, levels of the rarer, larger transcript were consistently reduced in a rapid, sustained, and dose-dependent manner following hydrogen peroxide treatment. Prior supplementation of the cells with beta carotene provided some protection against the reduction in levels of this transcript following hydrogen peroxide treatment. In contrast, vitamins C and E had no effect at the concentrations tested. We have now cloned the cDNA corresponding to this stress-responsive transcript and demonstrated that it is an incompletely processed product of the mitochondrial genome encompassing ATPase subunits 8 and 6 plus the adjacent gene for cytochrome c oxidase subunit 3. This decrease in one specific mitochondrial transcript may represent a novel mechanism for differential expression of mitochondrially-encoded genes.

The complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in South Africa.

Hepatitis C virus (HCV) genotype 5a is the predominant genotype in southern Africa with a high prevalence amongst infected blood donors from areas in South Africa. We have determined the nucleotide sequence corresponding to the complete coding region of an HCV isolate, EUH1480, previously classified as genotype 5a, from an Edinburgh haemophiliac. The sequence contained a single open reading frame (ORF) coding for a polyprotein of 3014 amino acids. Comparison with the polyprotein sequences from other HCV genotypes, where the ORF varies from 3008 to 3037 amino acids, showed the observed variation in size was due to differences in lengths of the envelope 2 and the nonstructural 5A proteins. The sequence divergence of HCV genotype 5 ranged from 29.4% nucleotide differences (24.91% amino acid differences) compared with genotype 1c to 32.5% nucleotide differences (30.3% amino acid differences) compared with 2a. Phylogenetic analysis of the available full length nucleotide sequences showed EUH1480 to form a branch distinct from the other HCV types, confirming the classification of type 5a as a separate genotype.

Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East.

Hepatitis C virus (HCV) type 4 is the predominant genotype found throughout the Middle East and parts of Africa, often in association with high population prevalence as in Egypt. To investigate more fully its evolutionary relationship with other genotypes of HCV, and to study its overall genome organization, we have determined the entire sequence encompassing the coding region of the genotype 4a isolate ED43, obtained from an HCV-infected individual from Egypt. The sequence of ED43 contained a single open reading frame encoding a polyprotein of 3008 amino acids (aa), smaller than that reported for other HCV genotypes which vary from 3010 aa to 3037 aa. The nucleotide and amino acid sequences were compared with the full-length sequences already reported for genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b and those of isolates JKO49 and JKO46 described as types 10a and 11a. The differences in length of the polyprotein originated in variable regions in the E2 and NS5A genes. The complete sequence of ED43 confirmed the classification of type 4 as a separate major genotype.

Complete coding sequence of hepatitis C virus genotype 6a.

Hepatitis C virus (HCV) genotype 6a is found in a restricted part of South East Asia, including Hong Kong, Macau and Vietnam. We determined the full length coding sequence of a type 6a isolate (EUHK2) obtained from a Hong Kong blood donor. The sequence of EUHK2 contained a single open reading frame coding for a polyprotein of 3018 amino acids, within the range of 3008 to 3037 for other HCV genotypes. The full length sequence of EUHK2 showed 30.3%-32.9% nucleotide (24.3%-29.4% amino acid) sequence divergence from genotypes 1-4, but only 27.7% (20.7% amino acid) divergence from JK046 ("type 11a"). These similarity values were intermediate between those of other HCV genotypes (minimum 28.4%) and between subtypes (maximum 25%). The close evolutionary relationship of EUHK2 with JK046 was further indicated by their grouping together by phylogenetic analysis.

Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm.

The Bunyamwera bunyavirus (BUN) M RNA genome segment encodes three proteins, two glycoproteins termed G1 and G2 and a non-structural protein called NSm, in the form of a polyprotein precursor that is co-translationally cleaved to give the mature proteins. Indirect immunofluorescence experiments have shown that these proteins localize to the Golgi complex in BUN-infected cells. We have used a recombinant vaccinia virus (vTF7-3), which expresses bacteriophage T7 RNA polymerase, to drive the expression of plasmids containing either the entire BUN M segment cDNA or fragments that encode the G1, G2 and NSm proteins separately under control of the T7 promoter. After transfection of these plasmids into vTF7-3-infected cells, correctly sized and processed proteins were detected by immunoprecipitation with BUN-specific antibodies. Immunofluorescence experiments showed that G1, G2 and NSm localized to the Golgi when transiently expressed from the full-length cDNA. When G2 or NSm were expressed separately they also localized to the Golgi, but when G1 was expressed alone a staining pattern typical for the endoplasmic reticulum was obtained. However coexpression of G2 and G1 from independent plasmids resulted in G1 localizing to the Golgi. In contrast translocation of G1 to the Golgi was not observed when G1 was coexpressed with NSm, although NSm itself was still detected in the Golgi. Similar results were obtained when the proteins were expressed from transfected plasmids containing the G2-, NSm- or G1-coding sequences under control of the cytomegalovirus immediate-early promoter. The localization of G1 to the Golgi when coexpressed with G2 was confirmed by the loss of endoglycosidase H (endo H) sensitivity of G1 after approximately 60 min in a pulse-chase experiment; G1 remained sensitive to endo H when expressed either alone or in combination with NSm. These results suggest that G2 contains the Golgi targeting and/or retention signals and that G1 has to interact with this protein to localize to this cellular compartment.

The S RNA genome segments of Batai, Cache Valley, Guaroa, Kairi, Lumbo, Main Drain and Northway bunyaviruses: sequence determination and analysis.

Bunyaviruses have a genome comprising three segments of negative-sense RNA. The smallest RNA segment, S, encodes the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. The sequences of the S genome RNA segments of seven bunyaviruses (Batai, Cache Valley, Guaroa, Kairi, Main Drain, Northway and Lumbo) were determined from cloned cDNAs obtained using a one-step reverse transcription-PCR protocol. These sequences were compared to those of six viruses previously published, reinforcing earlier conclusions about relationships of the bunyaviruses. Sequence homologies between N proteins correlated with the subdivision of these viruses into three serogroups, Bunyamwera, California and Simbu. The encoded N proteins are either 233 or 235 amino acids in length, depending on the serogroup, whereas the NSs proteins are more variable (83 to 109 amino acids). Certain nucleotide sequence motifs are conserved in the S segments of the Bunyamwera and California serogroup viruses, including the spacing of the AUG initiation codons for the N and NSs proteins (except Guaroa virus), and a CA-rich motif in the virion-sense RNA just downstream of the predicted mRNA termination site. A duplicated sequence was observed in the 3' non-coding region of the Lumbo virus S segment, which accounts for the significantly longer S genome segment of this virus.

Advanced use of tissue adhesive in hair transplantation.

BACKGROUND: Cobblestoning is an unsightly result of graft elevation in the recipient site after hair transplantation. OBJECTIVE: To describe the use of tissue adhesives for the avoidance of cobblestoning. RESULTS: The long-term cosmetic appearance of hair transplants may be improved through the use of cyanoacrylate adhesives is the virtual elimination of the common problem of graft elevation or cobblestoning, which produces an unsightly bumpiness in the recipient area. The choice of type of adhesive and the method of application are central to successful use of this technique. CONCLUSION: The indirect benefits of elimination of the need for postoperative bandages and restrictions in activities couple with the cosmetic results to produce a high degree of patient satisfaction.

Expression of the Bunyamwera virus M genome segment and intracellular localization of NSm.

Bunyamwera (BUN) virus is the prototype of the family Bunyaviridae and contains a trisegmented, single-stranded RNA genome of negative polarity. The medium (M) RNA segment encodes the two virion glycoproteins, G1 and G2, and a nonstructural protein, NSm, in the form of a polyprotein precursor which is cotranslationally cleaved. The gene order of the M segment is 5' G2-NSm-G1 3'. We have raised a monospecific antiserum in rabbits to a branched chain synthetic peptide to a region of the NSm protein which specifically immunoprecipitates NSm from BUN-infected cells. Indirect immunofluorescence experiments on BUN-infected cells using this antiserum gave a perinuclear staining pattern, suggesting that like the viral structural proteins, NSm localizes to the Golgi complex. An essentially full-length M segment cDNA was cloned into a recombinant vaccinia virus under control of bacteriophage T7 promoter and terminator sequences and expressed in cells co-infected with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. G1, G2, and NSm were detected in cells dually infected with the recombinant vaccina viruses, indicating that processing of the M segment-encoded precursor does not require other BUN proteins. Immunofluorescence experiments showed that the BUN glycoproteins expressed from this recombinant vaccinia virus system localized to the Golgi complex like authentic BUN proteins.