Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy.

There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression is typically performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes. Contamination of negative samples with such calibrants could cause false positive results. We developed a design strategy for synthetic reference materials (RMs) with modified transgenic sequences to prevent false positives due to cross-contamination. When such RM is amplified in transgene-specific assays, the amplicons are distinguishable from transgene's amplicons based on size and sequence. Using human erythropoietin as a model, we produced certified RM according to this strategy and following ISO Guide 35. Using non-viral and viral vectors, we validated the effectiveness of this RM in vector genome analysis in blood in vitro. The developed design strategy could be applied to production of RMs for other transgenes, genes or transcripts. Together with validated PCR assays, such RMs form a measurement tool that facilitates standardised, accurate and reliable genetic analysis in various applications.

Gene doping detection: evaluation of approach for direct detection of gene transfer using erythropoietin as a model system.

As clinical gene therapy has progressed toward realizing its potential, concern over misuse of the technology to enhance performance in athletes is growing. Although 'gene doping' is banned by the World Anti-Doping Agency, its detection remains a major challenge. In this study, we developed a methodology for direct detection of the transferred genetic material and evaluated its feasibility for gene doping detection in blood samples from athletes. Using erythropoietin (EPO) as a model gene and a simple in vitro system, we developed real-time PCR assays that target sequences within the transgene complementary DNA corresponding to exon/exon junctions. As these junctions are absent in the endogenous gene due to their interruption by introns, the approach allows detection of trace amounts of a transgene in a large background of the endogenous gene. Two developed assays and one commercial gene expression assay for EPO were validated. On the basis of ability of these assays to selectively amplify transgenic DNA and analysis of literature on testing of gene transfer in preclinical and clinical gene therapy, it is concluded that the developed approach would potentially be suitable to detect gene doping through gene transfer by analysis of small volumes of blood using regular out-of-competition testing.

Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis.

BACKGROUND AND OBJECTIVES: The detection of recombinant human erythropoietin (r-HuEPO) abuse by athletes remains problematic. The main aim of this study was to demonstrate that the five indirect markers of altered erythropoiesis identified in our earlier work were reliable evidence of current or recently discontinued r-HuEPO use. A subsidiary aim was to refine weightings of the five markers in the initial model using a much larger data set than in the pilot study. A final aim was to verify that the hematologic response to r-HuEPO did not differ between Caucasian and Asiatic subjects. DESIGN AND METHODS: Recreational athletes resident in Sydney, Australia (Sydney, n = 49; 16 women, 33 men) or Beijing, China (Beijing, n=24; 12 women, 12 men) were randomly assigned to r-HuEPO or placebo groups prior to a 25 day administration phase. Injections of r-HuEPO (or saline) were administered double-blind at a dose of 50 IU/kg three times per week, with oral iron (105 mg) or placebo supplements taken daily by all subjects. Blood profiles were monitored during and for 4 weeks after drug administration for hematocrit (Hct), reticulocyte hematocrit (RetHct), percent macrocytes (%Macro), serum erythropoietin (EPO) and soluble transferrin receptor (sTfr), since we had previously shown that these five variables were indicative of r-HuEPO use. RESULTS: The changes in Hct, RetHct, %Macro, EPO and sTfr in the Sydney trial were qualitatively very similar to the changes noted in our previous administration trial involving recreational athletes of similar genetic origin. Statistical models developed from Fisher's discriminant analysis were able to categorize the user and placebo groups correctly. The same hematologic response was demonstrated in Beijing athletes also administered r-HuEPO. INTERPRETATION AND CONCLUSIONS: This paper confirms that r-HuEPO administration causes a predictable and reproducible hematologic response. These markers are disturbed both during and for several weeks following r-HuEPO administration. This work establishes an indirect blood test which offers a useful means of detecting and deterring r-HuEPO abuse. Ethnicity did not influence the markers identified as being able to detect athletes who abuse r-HuEPO.

A novel method utilising markers of altered erythropoiesis for the detection of recombinant human erythropoietin abuse in athletes.

BACKGROUND AND OBJECTIVE: The use of recombinant human erythropoietin (r-HuEPO) to enhance athletic performance is prohibited. Existing tests cannot readily differentiate between exogenous and endogenous EPO. Therefore the aim of our study was to investigate possible indirect detection of r-HuEPO use via blood markers of altered erythropoiesis. DESIGN AND METHODS: Twenty-seven recreational athletes were assigned to three groups prior to a 25 day drug administration phase, with the following protocols: EPO+IM group (n = 10), 50 Ukg(-1) r-HuEPO at a frequency of 3wk(-1), 100 mg intramuscular (IM) iron 1wk(-1) and a sham iron tablet daily; EPO+OR group (n = 8), 50 U.kg(-1) r-HuEPO 3wk(-1), sham iron injection 1wk(-1) and 105 mg of oral elemental iron daily; placebo group (n = 9), sham r-HuEPO injections 3wk(-1), sham iron injections 1wk(-1) and sham iron tablets daily. Each group was monitored during and for 4 weeks after drug administration. RESULTS: Models incorporating combinations of the variables reticulocyte hematocrit (RetHct), serum EPO, soluble transferrin receptor, hematocrit (Hct) and % macrocytes were analyzed by logistic regression. One model (ON-model) repeatedly identified 94-100% of r-HuEPO group members during the final 2 wk of the r-HuEPO administration phase. One false positive was recorded from a possible 189. Another model (OFF-model) incorporating RetHct, EPO and Hct was applied during the wash-out phase and, during the period of 12-21 days after the last r-HuEPO injection, it repeatedly identified 67-72% of recent users with no false positives. INTERPRETATION AND CONCLUSIONS: Multiple indirect hematologic and biochemical markers used simultaneously are potentially effective for identifying current or recent users of r-HuEPO.

Simulated moderate altitude elevates serum erythropoietin but does not increase reticulocyte production in well-trained runners.

The purpose of this study was to investigate whether the modest increases in serum erythropoietin (sEpo) experienced after brief sojourns at simulated altitude are sufficient to stimulate reticulocyte production. Six well-trained middle-distance runners (HIGH, mean maximum oxygen uptake, VO2max = 70.2 ml x kg(-1) x min(-1) spent 8-11 h per night for 5 nights in a nitrogen house that simulated an altitude of 2650 m. Five squad members (CONTROL, mean VO2max= 68.9 ml x kg(-1) x min(-1) undertook the same training, which was conducted under near-sea-level conditions (600 m altitude), and slept in dormitory-style accommodation also at 600 m altitude. For both groups, this 5-night protocol was undertaken on three occasions, with a 3-night interim between successive exposures. Venous blood samples were measured for sEpo after 1 and 5 nights of hypoxia on each occasion. The percentage of reticulocytes was measured, along with a range of reticulocyte parameters that are sensitive to changes in erythropoiesis. Mean serum erythropoietin levels increased significantly (P < 0.01) above baseline values [mean (SD) 7.9 (2.4) mU x ml(-1)] in the HIGH group after the 1st night [11.8 (1.9) mU x ml(-1), 57%], and were also higher on the 5th night [10.7 (2.2) mU x ml(-1), 42%] compared with the CONTROL group, whose erythropoietin levels did not change. After athletes spent 3 nights at near sea level, the change in sEpo during subsequent hypoxic exposures was markedly attenuated (13% and -4% change during the second exposure; 26% and 14% change during the third exposure; 1st and 5th nights of each block, respectively). The increase in sEpo was insufficient to stimulate reticulocyte production at any time point. We conclude that when daily training loads are controlled, the modest increases in sEpo known to occur following brief exposure to a simulated altitude of 2650 m are insufficient to stimulate reticulocyte production.

Serotype classification and characterisation of the rotavirus SA11 VP6 protein using mass spectrometry and two-dimensional gel electrophoresis.

VP6, which makes up the inner capsid of rotavirus, is the major structural protein of this virus. Whilst VP6 has been sequenced at the DNA level in several rotavirus strains, there has been less effort to characterise the protein at the amino acid level. This paper reports the use of peptide mass fingerprinting and post-source decay fragmentation studies using MALDI-TOF and electrospray ionisation mass spectrometry to identify and characterise, in detail, the VP6 protein. We show that mass spectrometric analysis of VP6 peptides successfully distinguished SA11 from other rotavirus serotypes, and identify unique peptides that can be used for serotypic differentiation. For VP6 characterisation, the ExPASy FindMod tool was used to predict post-translational modifications on the protein. Analysis of trypsin and AspN digests predicted that the N-terminal methionine of VP6 was acetylated and this was confirmed using post source decay and electrospray ionisation mass spectrometry-mass spectrometry. An asparagine residue (aa107), which is followed by a glycine residue, was shown to undergo partial deamidation to aspartic acid. VP6 has two additional asparagine-glycine sequences and, in this sequence context, asparagine is known to be particularly susceptible to deamidation. Two-dimensional gel electrophoresis revealed a complex series of VP6 isoforms with an apparent molecular mass of approximately 45,000 Da and a pI ranging from 5.25 to 5.8. This pattern could partly be explained by the potential for deamidation at several sites within the protein.

Development of a rapid and sensitive quantitative assay for rotavirus based on flow cytometry.

A very sensitive and accurate flow cytometry (FC) based method have developed to quantitate rotavirus infection in MA104 cells. Confluent cell monolayers were infected with serial dilutions of rotavirus SA11. After infection, the cells were recovered with the aid of trypsin and then reacted with monoclonal antibody M60 (specific for the rotavirus outer capsid protein, VP7), followed by a second antibody (anti-mouse IgG-FITC). A FACScan FC was used to estimate the number of infected cells, as well as the level of infection. Viral infection was optimised by varying the concentration of trypsin used in the maintenance medium. The FC method enables many cells to be screened quickly for infectivity, and can detect low levels of virus. This method can be adapted to monitor the presence of other viruses in clinical and environmental samples without the need for prolonged periods of adaptation to growth in tissue culture.

Calcium influences the stability and conformation of rotavirus SA11 glycoprotein VP7 expressed in Dictyostelium discoideum.

We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca2+ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca2+ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca2+ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca2+ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca2+ and that conformational changes in VP7 which occur following depletion of Ca2+ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7.

From virus to vaccine: developments using the simple eukaryote, Dictyostelium discoideum.

Mass vaccination compaigns against viral diseases, both human and anim al, depend on the availability of cheap viral antigens. The eukaryote Dictyostelium discoideum has simple growth requirements and rapid growth rates and forms stable cell lines. These features, together with the possibility of secreting recombinant (glyco)proteins into a defined buffer, make the D. discoideum expression system an attractive option for producing economical recombinant subunit vaccines.

Recombinant glycoprotein production in the slime mould Dictyostelium discoideum.

Dictyostelium discoideum is a well known amoeboid organism, with unicellular and multicellular life-cycle stages, that is used for studying cell and developmental biology. With advances in gene-disruption technology and transformation of this organism, many homologous proteins have been expressed either to complement defective proteins or to study basic cell biology. Now, D. discoideum is being used to express heterologous proteins that are difficult to study in other systems, and its unique cell biology is being exploited to facilitate a wide range of protein modifications. In the past year, substantial progress has been made in expressing correctly folded forms of malarial circumsporozoite antigen and rotavirus surface glycoprotein VP7. Exciting developments have also been made in expressing human muscarinic receptors.

Expression of the rotavirus SA11 protein VP7 in the simple eukaryote Dictyostelium discoideum.

The outer capsid protein of rotavirus, VP7, is a major neutralization antigen and is considered a necessary component of any subunit vaccine developed against rotavirus infection. For this reason, significant effort has been directed towards producing recombinant VP7 that maintains the antigenic characteristics of the native molecule. Using a relatively new expression system, the simple eukaryote Dictyostelium discoideum, we have cloned the portion of simian rotavirus SA11 genome segment 9, encoding the mature VP7 protein, downstream of a native D. discoideum secretion signal sequence in a high-copy-number extrachromosomal vector. The majority of the recombinant VP7 expressed by transformants was intracellular and was detected by Western immunoblot following gel electrophoresis as two or three bands with an apparent molecular mass of 35.5 to 37.5 kDa. A small amount of VP7 having an apparent molecular mass of 37.5 kDa was secreted. Both the intracellular VP7 and the secreted VP7 were N glycosylated and sensitive to endoglycosidase H digestion. Under nonreducing electrophoresis conditions, over half the intracellular VP7 migrated as a monomer while the remainder migrated with an apparent molecular mass approximately twice that of the monomeric form. In an enzyme-linked immunosorbent assay, intracellular VP7 reacted with both nonneutralizing and neutralizing antibodies. The monoclonal antibody recognition pattern paralleled that found with VP7 expressed in either vaccinia virus or herpes simplex virus type 1 and confirms that D. discoideum-expressed VP7 is able to form the major neutralization domains present on viral VP7. Because D. discoideum cells are easy and cheap to grow, this expression system provides a valuable alternative for the large-scale production of recombinant VP7 protein.

Pathogenesis and treatment of acute hematogenous osteomyelitis: evaluation of current views with reference to an animal model.

Antibiotic therapy and surgical drainage of the bone are the most commonly accepted forms of therapy for acute hematogenous osteomyelitis; the efficacy of these two treatment modalities, however, has not been fully established. Treatment regimens remain empiric and open to much controversy. Many contentious issues concerning the management of this disease have arisen because the natural history of the untreated condition has not been properly understood, because therapeutic regimens alternative to those empirically initiated and perpetuated have not been adequately tested, and because most follow-up studies have dealt with small numbers of patients over relatively short periods. A highly reproducible animal model of acute hematogenous osteomyelitis was developed and utilized in a reexamination of issues regarding the natural history and pathology of the disease and the influence of appropriate treatment.

Isolation and characterisation of a family of small plasmids encoding resistance to nucleic acid-binding compounds in Staphylococcus aureus.

A family of small plasmids encoding resistance to nucleic acid-binding (NAB) compounds has recently been identified in strains of Staphylococcus aureus isolated in Italy, Texas and Western Australia. The mol. wts of the NAB-resistance plasmids are in the range (1.5-1.9) X 10(6) and all but one encode resistance to acridine yellow, ethidium bromide and quaternary ammonium compounds. The largest of the plasmids, pWG1773, differed in that it did not confer resistance to ethidium bromide. Restriction enzyme analysis of these plasmids revealed four distinct patterns corresponding to plasmids of four different mol. wts and physical maps were constructed based on the restriction patterns. Two plasmid types of molecular sizes approximately 2440 and 2240 base pairs had a 610-base pair region in common. Physical maps of the other two plasmid types were not related. The presence of a family of small NAB-resistance plasmids which carry no other known phenotypic markers provides further evidence for the strong selective advantage associated with maintenance of this determinant in clinical isolates of S. aureus.

Acute hematogenous staphylococcal osteomyelitis: the effects of surgical drilling and curettage in an animal model.

Surgical drilling and curettage of metaphyseal medullary bone were performed in chickens 4, 8 and 15 d after bacterial inoculation with Staphylococcus aureus producing osteomyelitis. Intramedullary drilling and curettage resulted in extensive vascular damage and necrosis of medullary bone but failed to remove all viable bacteria. An effective continuing drainage channel for the pus was not established due to the formation of blood clot within the drill hole. Surgery performed before the formation of a sequestrum resulted in further spread of bacteria within the medullary cavity and the formation of a larger sequestrum than that commonly observed in the bones of infected, untreated chickens. Surgery performed after the formation of a sequestrum destroyed the abscess wall thus disrupting a natural defence mechanism of the host. Whilst a direct comparison of surgical drilling in human osteomyelitis and in this avian model cannot be made, the results suggest that production of an artificial drill hole across the cortex and curettage of the bone in human osteomyelitis would be destructive and may not be warranted.

A resistance determinant to nucleic acid-binding compounds in methicillin-resistant Staphylococcus aureus.

Recent isolates of methicillin-resistant Staphylococcus aureus from Australia and several other countries carry a plasmid coding for high levels of resistance to propamidine isethionate and low levels of resistance to cetyltrimethyl-ammonium bromide. The reasons for the acquisition and retention of such a determinant are not known. To define the properties of this resistance determinant in more detail, the minimum inhibitory concentrations of a series of cationic agents, including ethidium bromide, were determined and a cationic-resistance profile prepared for several strains of S. aureus and their isogenic, sensitive derivatives. The compounds for which resistance is coded by this determinant all bind to nucleic acids. Hence the determinant has been designated the NAB (nucleic acid-binding compounds)-resistance determinant.

Acute hematogenous staphylococcal osteomyelitis: evaluation of cloxacillin therapy in an animal model.

A reproducible avian model of acute hematogenous staphylococcal osteomyelitis was used to investigate various aspects of antibiotic therapy using a single antibiotic, cloxacillin. The effects of both delaying antibiotic administration and increasing the frequency of antibiotic administration on the outcome of the disease were evaluated. Following bacterial inoculation, a delay in commencing therapy for 4 d in comparison to 1 d significantly reduced the likelihood of a favourable outcome. An increase in the frequency of antibiotic administration from once to 4 times daily resulted in significant improvement in the recovery rate of chickens. Whilst repeated antibiotic administration controlled the systemic effects of the disease and improved the clinical status of chickens, the local bone lesion was not always sterilized. The role of antibiotic therapy in acute hematogenous osteomyelitis is discussed in relation to these findings.

Acute haematogenous osteomyelitis: II. The effect of a metaphyseal abscess on the surrounding blood supply.

Three major vessel types--metaphyseal, epiphyseal and transphyseal vessels--were identified in the region of the growth plate following perfusion of the vasculature of the chicken leg with a solution of Berlin Blue. Following the production of osteomyelitis, obstruction to the blood supply adjacent to the abscess was rapid and was clearly demonstrated due to a lack of perfusion of the involved vessels with Berlin Blue. The extent of disturbance to the blood supply was dependent on the type of vessel involved in the inflammatory process. The efficacy of bloodstream therapy is discussed in relation to these findings.

Early functional and morphological changes in renal tubular necrosis due to p-aminophenol.

Functional and morphological changes developed rapidly in rats after the intravenous administration of the organic nephrotoxin p-aminophenol. Proximal intratubular pressure remained close to its mean control value of 14.9 +/- 0.9 mm Hg up to 40 min after injection of the nephrotoxin then rose rapidly over the following 50 min to a maximum of 38.7 +/- 7.4 mm Hg. Distal tubular pressure also rose in the same manner. Renal blood flow remained constant, but GFR fell to 11% of control values while fractional excretion of sodium and water rose 12 and five times, respectively. Morphological changes developed in parallel with the functional changes. They were widespread, varied in intensity from cell to cell, were more severe in the distal third of the proximal convoluted tubule and consisted of cytoplasmic swelling, reduced organelle concentration, reduction or loss of basal infoldings, widening of lateral intercellular spaces, extrusion of bubbles of cell sap into the tubular lumen; brush borders were preserved. No casts were present up to 90 min. Similar results were seen when p-aminophenol was added to the perfusate of the isolated perfused kidney. It is proposed that metabolic and morphological damage leads rapidly to both impairment of proximal tubular sodium reabsorption and increased flow resistance in the cortical collecting system. Both effects contribute to a rise in tubular pressures which oppose glomerular filtration.

Acute haematogenous osteomyelitis: an experimental model.

A simple and reproducible model of acute haematogenous staphylococcal osteomyelitis is described. Twenty-nine day-old chickens were inoculated intravenously with 10(4)-10(8) viable organisms Staphylococcus aureus per kg body weight and were killed 1-8 days after inoculation. Macroscopic septic foci could be detected within 24 hr of inoculation and were most commonly situated in the metaphyseal region of the proximal tibia and distal femur. Lesions in other organs were not observed. The production of osteomyelitis was dependent on the bacterial inoculum size. It was estimated that 5.5 X 10(5) viable organisms per kg body weight of chicken were required to produce osteomyelitis in 50 per cent of injected chickens. Chicken weights were monitored throughout the experiment. A close negative correlation existed between the logarithm of the bacterial inoculum size and the chicken growth rate in the first 24 hr following inoculation (r = -0.968, P less than 0.01). The chicken growth rate was therefore used as an accurate predictor of osteomyelitis in individual chickens.