Catheter length preference in wheelchair-using men who perform routine clean intermittent catheterization.

STUDY DESIGN: Prospective, unblinded, multicenter, randomized, controlled, cross-over study assessing user preference and ease of use characteristics of two gel intermittent catheters in 81 self-catheterizing wheelchair-using men. OBJECTIVES: To evaluate the male user's preference between a 30-cm and a 40-cm intermittent catheter (Apogee Intermittent Catheter, Hollister Incorporated, Libertyville, IL, USA) regarding the ease of insertion and removal, ability to control the catheter during insertion, bladder emptying confidence and ease of draining urine into a receptacle or connecting to a urine bag. SETTING: Multiple institutions in the United States. METHODS: Subjects were randomized to order of catheter use, using both 10 test catheters (30-cm) and 10 control catheters (40 cm). All catheters were 12 or 14 French and identical in design and composition, except length. Safety was assessed during the entire study period regarding adverse events (AE) and adverse device events (ADE). Subjects evaluated their ease of use characteristics after each catheter use and final catheter preference. RESULTS: Subjects preferred the Apogee 40-cm intermittent catheter (91.4%) over the 30-cm length (8.6%). The preference was due to subject confidence of complete bladder emptying (70%), more satisfactory length (74%) and easier to drain into a receptacle (58%) with a portable urinal being the most utilized (37%), followed by toilet (35%). The only AE/ADE reported was minor urethral bleeding in one subject and minor pain in another subject, both with the 30-cm catheter. None were reported with the 40-cm catheter. CONCLUSIONS: The Apogee 40-cm catheter was the preferred intermittent catheter due to subject confidence in bladder emptying, ease of catheter manipulation and the ease of draining urine into a receptacle.

Role of genetically engineered animals in future food production.

Genetically engineered (GE) animals are likely to have an important role in the future in meeting the food demand of a burgeoning global population. There have already been many notable achievements using this technology in livestock, poultry and aquatic species. In particular, the use of RNA interference (RNAi) to produce virus-resistant animals is a rapidly-developing area of research. However, despite the promise of this technology, very few GE animals have been commercialised. This review aims to provide information so that veterinarians and animal health scientists are better able to participate in the debate on GE animals.

Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino.

We have earlier shown that antisense morpholino oligomers are able to restore dystrophin expression by systemic delivery in body-wide skeletal muscles of dystrophic mdx mice. However, the levels of dystrophin expression vary considerably and, more importantly, no dystrophin expression has been achieved in cardiac muscle. In this study, we investigate the efficiency of morpholino-induced exon skipping in cardiomyoblasts and myocytes in vitro, and in cardiac muscle in vivo by dose escalation. We showed that morpholino induces targeted exon skipping equally effectively in both skeletal muscle myoblasts and cardiomyoblasts. Effective exon skipping was achieved in cardiomyocytes in culture. In the mdx mice, morpholino rescues dystrophin expression dose dependently in both skeletal and cardiac muscles. Therapeutic levels of dystrophin were achieved in cardiac muscle albeit at higher doses than in skeletal muscles. Up to 50 and 30% normal levels of dystrophin were induced by single systemic delivery of 3 g kg(-1) of morpholino in skeletal and cardiac muscles, respectively. High doses of morpholino treatment reduced the serum levels of creatine kinase without clear toxicity. These findings suggest that effective rescue of dystrophin in cardiac muscles can be achieved by morpholino for the treatment of Duchenne muscular dystrophy.

Microwave irradiation enhances gene and oligonucleotide delivery and induces effective exon skipping in myoblasts.

Microwave (MW) energy consists of electric and magnetic fields and is able to penetrate deep into biological materials. We investigated the effect of MW (2450 MHz) irradiation on gene delivery in cultured mouse myoblasts and observed enhanced transgene expression. This effect is, however, highly variable and critically dependent on the power levels, duration and cycle conditions of MW exposure. MW irradiation greatly enhances delivery of 2'O methyl-phosphorothioate antisense oligonucleotide (AON) targeting mouse dystrophin exon 23 and induces specific exon skipping in cultured myoblasts. Effective delivery of AON by MW irradiation is able to correct the dystrophin reading frame disrupted by a nonsense point mutation in the H2K mdx myoblasts, resulting in the restoration of dystrophin expression. MW-mediated nucleic acid delivery does not directly link to the increase in system temperature. The high variability in gene and oligonucleotide delivery is most likely the result of considerable irregularity in the distribution of the energy and magnetic field produced by MW with the current device. Therefore, achieving effective delivery of the therapeutic molecules would require new designs of MW devices capable of providing controllable and evenly distributed energy for homogenous exposure of the target cells.

The use of RNAi and transgenics to develop viral disease resistant livestock.

The possibility of genetically engineering poultry to make them resistant to avian influenza is attracting attention and has now become a real possibility with improved methods for genetic modification and the emergence of RNAi as an antiviral strategy. In order to test this possibility, we have generated transgenic mice that express RNAi molecules targeting a conserved region of the influenza A NP gene and are testing these mice for resistance to influenza infection. Transgenes were initially developed that express short hairpin RNAs (shRNAs) targeting multiple influenza A viral genes. The shRNAs were tested for inhibition of H1N1 PR8 virus in vitro. Two potent shRNAs that target the NP and PA genes were chosen for lentiviral mediated generation of transgenic mice. Transgenic founders for the NP shRNA construct and also a negative control shRNAtargeting EGFP were generated. The constitutive expression of the shRNA molecules in a range of tissue types including lung, was confirmed and so far stable transmission of the RNAi transgenes from the F0 to F3 generation has been observed. Resistance to influenza infection in these transgenic mice is now being confirmed.

Manipulation of small RNAs to modify the chicken transcriptome and enhance productivity traits.

In recent years there has been a revolution in our understanding of genes and how they come to control the physical outcomes of development. Central to this has been the understanding of the cellular processes of RNA interference (RNAi), for which the Nobel Prize for Physiology or Medicine was awarded in 2006. Coupled with this has been the recognition that microRNAs are key mediators of this process within cells. RNAi whether mediated exogenously by synthetic oligonucleotides or vector-delivered double stranded RNA or endogenously by microRNAs can have a profound and specific effect on gene expression. Elucidating and understanding these processes in the chicken will provide critical information to enable more precise control over breeding strategies for improvement of traits in production poultry, either by direct or indirect means. It will also provide alternative strategies for the control and prevention of important avian diseases.

Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression.

RNA interference (RNAi) mediated by DNA-based expression of short hairpin RNA (shRNA) is a powerful method of sequence-specific gene knockdown. A number of vectors for expression of shRNA have been developed that feature promoters from RNA polymerase III (pol III)-transcribed genes of mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and for future development of specific therapeutics in the bovine species, we have developed shRNA expression vectors that feature novel bovine RNA pol III promoters. We characterized two bovine U6 small nuclear RNA (snRNA) promoters (bU6-2 and bU6-3) and a bovine 7SK snRNA promoter (b7SK). We compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and suppressing the reporter gene encoding enhanced green fluorescent protein. Results show that the b7SK promoter induced the greatest level of suppression in a range of cell lines. The comparison of these bovine promoters in shRNA expression is an important component for the future development of bovine-specific RNAi-based research.

Improved vectors for expression library immunization--application to Mycoplasma hyopneumoniae infection in pigs.

Expression library immunization (ELI) has previously been used in a number of disease models in mice. Here, we describe the first example of the application of ELI to a large animal model with the immunization of pigs against enzootic pneumonia, a disease caused by Mycoplasma hyopneumoniae. The development of new plasmid vectors and library screening methods facilitated the application of ELI to this disease by allowing random libraries to be screened for clones expressing recombinant proteins. In this way the vast majority of clones in random libraries that are unproductive can be eliminated, meaning that libraries are more likely to give protection and are subsequently easier to further screen and analyze. By using this approach we have used one library screen and two animal trials to progress from an original library of 20,000 clones to a group of just 96 clones.

A large, single center investigation of the immunogenetic factors affecting liver transplantation.

BACKGROUND: Reports on the relevance of immunogenetic factors in liver transplantation are often conflicting or inconclusive. We have, therefore, investigated a range of factors that may underlie liver graft survival. METHODS: The influences of HLA, flow cytometric, and enhanced cytotoxic crossmatching and immunoglobulin (Ig)A levels on graft survival, and acute and chronic rejection were investigated for a single center involving 446 patients over 13 years. RESULTS: The effect of HLA mismatching on graft survival was significant (P<10(-2)) and was reversed in recipients with autoimmune diseases (P<0.5x10(-2)), whereas the effect of HLA mismatches on the level of acute rejection was detrimental in all recipients. There was a significant effect of a positive cytotoxic crossmatch on 3-month (P<10(-5)) and 1-year (P<10(-4)) graft survival, and an additional effect of the flow cytometric crossmatch was seen for chronic rejection (P<10(-2)) and acute rejection (P<10(-2)). Recipients with HLA-A1,B8,DRB1*0301 had higher levels of acute rejection (P<0.5x10(-2)), and recipients who received an ABO compatible-nonidentical transplant have a significantly higher risk (P<10(-2)) of developing chronic rejection. Finally, the beneficial effect of high serum IgA and, specifically, IgA anti Fab, seen in renal transplants was not evident in liver transplants, and in fact the opposite may be true, at least for acute rejection (P<0.5x10(-2)). CONCLUSIONS: By separating the recipients with autoimmune disease from other patients and by including acute and chronic rejection as outcome parameters, we have used the power of a large single-centre study to delineate the significance of some of the important immunogenetic factors involved in liver transplantation.

IgA class antibodies and flow cytometric cross-matching in renal transplantation.

BACKGROUND: The established method of pretransplant cross-matching does not detect IgA antibodies, and IgA antibodies have thus been ignored when assessing patients for transplantation. The aim of this study was to detect IgA allo- and autoreactive antibodies using flow cytometry and to correlate the results with transplant outcome. METHODS: Pretransplant sera from 231 sequential renal recipients were tested for serum IgA levels and antibodies directed against the Fab portion of the human IgG molecule. Fifty-nine recipients with sufficient stored donor lymphocytes were also tested by flow cytometry for donor-specific alloantibodies of the IgA isotype. RESULTS: Graft survival was improved in recipients with higher IgA levels. High IgA anti-Fab levels led to a significantly higher 1-year graft survival (P<0.05). Graft survival was further enhanced where both serum IgA and IgA anti-Fab were raised (P<0.01). Although the mean IgA level tended to be higher for recipients with a positive IgA flow cytometric cross-match (FCXM), the IgA FCXM was not associated with increased IgA anti-Fab, suggesting that the IgA FCXM is detecting a different subset of IgA reactivity. Additionally, for primary grafts, a positive IgA FCXM was not associated with enhanced graft survival. CONCLUSIONS: Within the repertoire of IgA activity, there are two recognizable groups, the IgA anti-Fab specificity, which is significantly associated with enhanced graft survival, and that detected by the IgA FCXM, which surprisingly is more likely to be positive in less sensitized first grafts and is not associated with enhanced graft survival.

Putative functional domain within ORF2 on the Mycobacterium insertion sequences IS900 and IS902.

Repeated DNA sequences have been identified in a range of mycobacterial species and have been implicated in the increased virulence of some of these species, namely, Mycobacterium paratuberculosis and M. avium subsp. silvaticum. Here we present a case to suggest that the insertion sequences IS900 and IS902 encode a protein from a putative gene positioned on the complementary strand to their transposase genes. Based on amino acid homology analyses, this open reading frame (ORF2) could encode a transport protein. The ORF2 protein thus IS900 and IS902, may have a role in the increased pathogenicity of M. paratuberculosis and M. avium subsp. silvaticum from an M. avium background.

Molecular analysis of the F plasmid traVR region: traV encodes a lipoprotein.

The nucleotide sequences of the conjugative F plasmid transfer region genes, traV and traR, have been determined. The deduced amino acid sequence of TraV indicated that it may be a lipoprotein; this was confirmed by examining the effect of globomycin on traV-encoded polypeptides synthesized in minicells. An open reading frame that may represent a previously undetected transfer gene, now designated trbG, was identified immediately upstream of traV. The deduced product of traR was found to share amino acid similarity with proteins from the bacteriophages 186 and P2 and with the dosage-dependent dnaK suppressor DksA.

Characterisation of a highly repeated DNA sequence from Mycobacterium bovis.

We report characterisation of a novel repeat sequence from a Mycobacterium bovis genomic library. The highly repeated sequence belongs to a family consisting of a 24 base pair (bp) direct repeat (DR), that appears to be organized into clusters on the chromosome. We classify the 24-bp DR into the group of prokaryotic DNA repeats known as the interspersed repetitive sequence elements. The 24-bp DR will be of potential use as a DNA fingerprinting tool in epidemiological studies of M. bovis.

Restriction fragment length polymorphism analysis of Fusobacterium necrophorum using a novel repeat DNA sequence and a 16S rRNA gene probe.

A repeated DNA sequence was isolated from Fusobacterium necrophorum biotype AB, strain FnS1. The repeated sequence shared considerable homology with the transposase gene from the Pseudomonas syringiae insertion sequence IS801. The repeat sequence was used together with a 16S ribosomal RNA gene probe to type F. necrophorum isolates using restriction fragment length polymorphisms. The probes revealed differences between several clinical isolates and will be useful tools to study the epidemiology of ovine foot abscess and other diseases caused by F. necrophorum.

Treatment of recurrent spontaneous abortion by immunization with paternal lymphocytes: results of a controlled trial.

PROBLEM: It remains unclear whether maternal immunization with paternal lymphocytes prior to conception improves the reproductive outcome in women with recurrent abortion in whom all secondary causes have been excluded. METHOD: A double-blind placebo controlled trial was instituted in women with unexplained recurrent spontaneous abortion, comparing immunization with 400 million paternal to 400 million maternal (autologous) lymphocytes. The groups were compared in a paired sequential trials chart, by logistic regression, and, in addition, a meta-analysis of this and other published trials was carried out. RESULTS: The live birth rate among pregnancies in paired couples with paternal lymphocyte immunization was 68% compared to 47% in the women who received their own cells. The results bordered on, but did not achieve, statistical significance. The women in each group were thoroughly investigated to exclude known causes of recurrent pregnancy loss and appeared to have been well matched in all variables. Women with lymphocytotoxic antibodies against paternal lymphocytes were excluded. Unlike our previous study there was not association between the time to conception and the chance of a successful outcome. Indeed, the time to conception was relatively short, 12 wk in all groups. The meta-analysis supported an overall modest favorable experience with paternal cells. CONCLUSION: The study is consistent with a general trend favoring paternal over maternal lymphocyte immunization but reinforces the need for larger multicenter controlled trials as well as more detailed biological study in humans to understand the nature of the maternal-fetal interface and its breakdown.

Characterisation of a novel repetitive DNA sequence from Mycobacterium bovis.

We report characterisation of three copies of a novel repeat sequence isolated from a Mycobacterium bovis genomic library. The repeat occurs within open reading frames, potentially encoding a conserved tandem array of a pentapeptide sequence with the consensus X-Gly-Asn-X-Gly. The tandem array is present up to five times in M. bovis and it is proposed that they may occur in a family of genes expressing functionally related proteins. We postulate that these proteins may play a role in binding of M. bovis to host cell receptors.

Patients with thick melanomas surviving at least 10 years: histological, cytometric and HLA analyses.

Survival for melanoma patients with thick primary tumours is notoriously short. A small number of patients with tumours greater than 5.5 mm thick do, however, have protracted survival intervals. Attempts were made to account for this phenomenon by means of histological, cytometric and HLA serotyping analyses. Patients with thick lesions surviving more than 10 years were matched--by sex, age, anatomical site of primary lesion, stage of disease and, whenever possible, by initial surgical therapy--to patients dying of their disease within 5 years. This case-control study on 13 long-term survivors and 13 short-term survivors did not show that any of the following attributes of the primary lesion were useful in predicting survival: Clark's level of invasion, ulceration, mitotic rate, host inflammatory response, tumour regression, tumour necrosis, vascular invasion, satellitosis, radial or vertical growth phase, predominant cell type, histogenetic type, borders, DNA quantification and cytomorphometry. HLA serotyping of long-term survivors showed an excess of antigen DQw1 compared with the general population, although this excess was not statistically significant.

Amino acid sequences recognized by T cells: studies on a merozoite surface antigen from the FCQ-27/PNG isolate of Plasmodium falciparum.

Twenty-six overlapping peptides, spanning the entire FCQ-27/PNG sequence of the Plasmodium falciparum antigen known as merozoite surface antigen 2 were screened for their ability to induce the proliferation of peripheral blood lymphocytes (PBL) obtained from 12 donors living in Honiara, Solomon Islands where P. falciparum is endemic. A recombinant (r) form of MSA2, known as Ag 1609 was also screened in these assays and tetanus toxoid (TT) antigen was included as a control. The location of the predicted T cell determinants within MSA2 was examined using the algorithm, AMPHI and by scanning MSA2 for amino acid sequences showing the Rothbard motif. There were 13 predicted amphipathic helical sites and five examples of Rothbard sequences in the antigen. The location of these with regard to the peptides tested is shown. Nine of the 12 individuals responded to TT with high stimulation indices (greater than 4) being obtained in the majority of donors. Only three individuals responded to r-MSA2 with the stimulation indices (SI) in the range of 2.4-4.1. Peptides from both the constant and variable regions of MSA2 were recognized in the proliferative assays. However, the majority of the positive proliferative responses were to peptides which spanned the central variable region which included the two copies of the 32-amino-acid repeat occurring in the antigen. High SI comparable to those obtained to TT were seen in some individuals with some peptides. There was considerable variation between donors in number and nature of the peptides recognised and two donors did not respond to any of the antigens tested. The significance of these findings to vaccine development is discussed.