Quantifying the effect of urodynamic catheters on urine flow rate measurement.

INTRODUCTION: The effect of urodynamic catheters on urine flow rate (Q(max) ) is well documented but under-researched. Several studies show reduced Q(max) but methodologies and patient demographics differ. The aims of this study were to further quantify the effect of urodynamic catheters on Q(max) and to explore if this was consistent across different urodynamic diagnoses. METHODS: Four groups of 50 consecutive men attending for urodynamic studies (UDS) were retrospectively analyzed: Group 1 comprised 50 men with normal UDS, Group 2 was 50 men with BOO, and Group 3 contained 50 men with detrusor underactivity. Groups 1-3 had UDS performed using both 10 Fr filling and 4 Fr measuring catheters in situ. Group 4 comprised 50 men who had UDS performed with a smaller catheter assembly (8 Fr dual-lumen). Values of Q(max) with and without catheters present were compared using paired Student's t-tests. Differences between groups were compared using ANOVA. RESULTS: Q(max) measured during UDS in men from Groups 1-3 showed a mean reduction of 38% compared to Q(max) from "free" uroflowmetry. ANOVA indicated this reduction was significantly greater among men with normal UDS. Interestingly the group who underwent UDS with a smaller catheter assembly showed no significant reduction in Q(max) measured with catheters in situ. CONCLUSION: Our findings are in line with previous work suggesting that smaller calibre urethral catheters do not cause a significant obstructive effect during voiding. In addition it would appear that the reduction in Q(max) with larger urethral catheters in situ is greatest in those with normal urodynamics.

Effects of preexisting immunity on the response to herpes simplex-based oncolytic viral therapy.

Herpes simplex viruses (HSV) type 1 are the basis of a number of anticancer strategies that have proven efficacious in animal models. They are natural human pathogens and the majority of adults have anti-HSV immunity. The current study examined the effect of preexisting immunity on the response to herpes-based oncolytic viral treatment of hepatic metastatic cancer in a murine model designed to simulate a clinical approach likely to be utilized for nonneurological tumors. Specifically, the anticancer effects of NV1020 or G207, two multimutated HSV-1 oncolytic viruses, were tested in immunocompetent mice previously immunized with a wild-type herpes simplex type 1 virus. Mice were documented to have humoral as well as cell-mediated immunity to HSV-1. Tumor response to oncolytic therapy was not measurably abrogated by immunity to HSV at the doses tested. The influence of route of viral administration was also tested in models of regional hepatic arterial and intravenous therapy. Route of viral administration influenced efficacy, as virus delivered intravenously produced some detectable attenuation while hepatic arterial therapy remained unaffected. These results demonstrate that when given at appropriate doses and in reasonable proximity to tumor targets, HSV-based oncolytic therapy can still be expected to be effective treatment for patients with hepatic malignancies.

Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genomes.

Herpes simplex virus (HSV) is a new platform for gene therapy. We cloned the human herpesvirus HSV-1 strain F genome into a bacterial artificial chromosome (BAC) and adapted chromosomal gene replacement technology to manipulate the viral genome. This technology exploits the power of bacterial genetics and permits generation of recombinant viruses in as few as 7 days. We utilized this technology to delete the viral packaging/cleavage (pac) sites from HSV-BAC. HSV-BAC DNA is stable in bacteria and the pac-deleted HSV-BAC (p45-25) is able to package amplicon plasmid DNA as efficiently as a comparable pac-deleted HSV cosmid set when transfected into mammalian cells. Moreover, the utility of bacterial gene replacement is not limited to HSV, since most herpesviruses can be cloned as BACs. Thus, this technology will greatly facilitate genetic manipulation of all herpesviruses for their use as research tools or as vectors in gene therapy.

Recurrent acyclovir-resistant herpes simplex in an immunocompromised patient: can strain differences compensate for loss of thymidine kinase in pathogenesis?

To investigate how acyclovir-resistant (ACVr) herpes simplex virus (HSV) evades drug therapy and causes disease, HSV-1 isolates from a bone marrow transplant (BMT) patient were studied. The patient developed ACVr disease after an initial BMT and, following a second BMT, reactivated ACVr HSV despite high-dose acyclovir prophylaxis. ACVr isolates from each episode contained the same point mutation in the viral thymidine kinase (tk) gene, documenting the emergence, latency, and reactivation of this mutant. The mutants were exceedingly impaired for TK activity in sensitive enzyme, plaque autoradiography, and drug-susceptibility assays. Nevertheless, these mutants and a tk deletion mutant constructed in the same genetic background reactivated from latency in mouse trigeminal ganglia, in contrast to similar mutants from laboratory strains. It is hypothesized that alleles in the clinical isolate compensate for the loss of TK in this animal model. Such genetic variability may be important for ACVr disease in humans.

Translational recoding induced by G-rich mRNA sequences that form unusual structures.

We investigated a herpesvirus mutant that contains a single base insertion in its thymidine kinase (tk) gene yet expresses low levels of TK via a net +1 translational recoding event. Within this mutant gene, we defined a G-rich signal that is sufficient to induce recoding. Unlike other translational recoding events, downstream RNA structures or termination codons did not stimulate recoding, and paused ribosomes were not detected. Mutational analysis indicated that specific tRNAs or codon-anticodon slippage were unlikely to account for recoding. Rather, recoding efficiency correlated with the G-richness of the signal and its ability to form unusual structures. These findings identify a mechanism of translational recoding with unique features and potential implications for clinical drug resistance and other biological systems.

Cloning, sequencing and expression of the S protein gene from two geographically distinct strains of canine coronavirus.

The gene encoding the spike (S) protein from two geographically distinct strains (American and British) of canine coronavirus (CCV) was cloned and sequenced. The nucleotide sequence revealed open reading frames of 1443 or 1453 amino acids, respectively. Structural features include an N-terminal hydrophobic signal sequence, a hydrophilic cysteine-rich cluster near the C-terminus, two heptad repeats and 29 or 33 potential N-glycosylation sites. Pairwise comparisons of S amino acid sequences from these isolates with other CCV strains (Insavc1 and K378) revealed that heterogeneity, found mostly in the form of conservative substitutions, is distributed throughout the canine sequences. However, 5 variable regions could be identified. Similar analysis with feline, porcine, murine, chicken and human coronavirus sequences revealed that the canine sequences are much more closely related to the feline S protein sequence than to the porcine S protein sequences even though they are all from the same antigenic group. Moreover, the sequence similarity between CCV isolates and the feline coronavirus, feline infectious peritonitis virus (FIPV) was comparable. Expression of the CCV or the transmissible gastroenteritis virus (TGEV) S gene using the vaccinia virus system produced a protein of the expected size which could induce extensive syncytia formation in infected canine A72 cells.

A net +1 frameshift permits synthesis of thymidine kinase from a drug-resistant herpes simplex virus mutant.

Clinical resistance to antiviral drugs requires that a virus evade drug therapy yet retain pathogenicity. Thymidine kinase (TK)-negative mutants of herpes simplex virus are resistant to the drug, acyclovir, but are attenuated for pathogenicity in animal models. However, numerous cases of clinical resistance to acyclovir have been associated with viruses that were reported to express no TK activity. We studied an acyclovir-resistant clinical mutant that contains a single-base insertion in its tk gene, predicting the synthesis of a truncated TK polypeptide with no TK activity. Nevertheless, the mutant retained some TK activity and the ability to reactivate from latent infections of mouse trigeminal ganglia. The mutant expressed both the predicted truncated polypeptide and a low level of a polypeptide that comigrated with full-length TK on polyacrylamide gels and reacted with anti-TK antiserum, providing evidence for a frameshifting mechanism. In vitro transcription and translation of mutant tk genes, including constructs in which reporter epitopes could be expressed only if frameshifting occurred, also gave rise to truncated and full-length polypeptides. Reverse transcriptase-polymerase chain reaction analysis coupled with open reading frame cloning failed to detect alterations in tk transcripts that could account for the synthesis of full-length polypeptide. Thus, synthesis of full-length TK was due to an unusual net +1 frameshift during translation, a phenomenon hitherto confined in eukaryotic cells to certain RNA viruses and retrotransposons. Utilization of cellular frameshifting mechanisms may permit an otherwise TK-negative virus to exhibit clinical acyclovir resistance.

Basophil leucocytes in responses to parasitic infection and some other stimuli in sheep.

Basophil leucocytes are a significant component of the infiltrating cells in a variety of tissue reactions in guinea pigs. However, little is known about the participation of basophils in similar reactions in most other animal species. The circulating blood, skin and small intestinal mucosa of sheep were examined after they had received stimuli known to elicit basophil-rich responses in guinea pigs but relatively few basophils were found.

Infectious crystalline keratopathy.

Two cases of typically appearing crystalline keratopathy both grew an alpha haemolytic streptococcus and had many of the features common to other cases. In one case, the use of topical and systemic antibiotics alone resulted in resolution of the disease, while in the second case even after corneal transplantation, a recurrence occurred which required further long-term antibiotics plus a repeat transplant.

Relationship between eosinophilia and responsiveness to infection with Trichostrongylus colubriformis in sheep.

Lambs with genetically determined increased immunological responsiveness to Trichostrongylus colubriformis (high responders) had more eosinophils in cutaneous reactions to the mitogen phytohaemagglutinin (PHA) both before and during infection compared with those bred for susceptibility (low responders). In contrast, eosinophil numbers in both blood and cutaneous reactions elicited by third-stage T. colubriformis larval antigen were similar in high and low responders before infection. Following vaccination and challenge, high responders had elevated eosinophil numbers in blood and antigen-stimulated skin. In unselected sheep, although eosinophil numbers in cutaneous reactions to PHA were related to responsiveness to a challenge infection with T. colubriformis, there was a closer relationship between blood eosinophil numbers and responsiveness. Infection with T. colubriformis increased eosinophil numbers in cutaneous reactions to PHA and appeared to augment the difference between eosinophil counts in high and low responder sheep. Measurement of the ability to produce eosinophil activating factors, or for eosinophils to respond to such factors might therefore be useful in identifying individual sheep with increased responsiveness to T. colubriformis infection.

Ocular injuries caused by magpies.

This paper presents a series of six patients with ocular injuries resulting from magpie attacks. Five cases involved children. In two cases the penetration was overlooked initially. In one case the keratitis was caused by Bacillus cereus. Full ophthalmic examination, including indirect ophthalmoscopy and microbiological studies, must be undertaken initially to identify unrecognised eye injuries and to prevent the possible sight-threatening complications of vitreal fibrosis with subsequent retinal detachment or endophthalmitis.

Analysis of a 9.6 kb sequence from the 3' end of canine coronavirus genomic RNA.

We have analysed the organization of the 3' end of the genomic RNA of canine coronavirus (CCV), a virus which has a close antigenic relationship to transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV) and feline infectious peritonitis virus (FIPV). Genomic RNA isolated from CCV strain Insavc-1-infected A72 cells was used to generate a cDNA library. Overlapping clones, spanning approximately 9.6 kb [from the 3' end of the polymerase gene, 1b, to the poly(A) tail] were identified. Sequencing and subsequent analyses revealed 10 open reading frames (ORFs). Three of these code for the major coronavirus structural polypeptides S, M and N; a fourth codes for a small membrane protein, SM, a putative homologue of the IBV structural polypeptide 3c, and five code for polypeptides, designated 1b, 3a, 4, 7a and 7b, homologous to putative non-structural polypeptides encoded in the TGEV or FIPV genomes. An extra ORF which had not hitherto been identified in this antigenic group of coronaviruses was designated 3x. Pairwise alignment of these ORFs with their counterparts in TGEV, PRCV and FIPV revealed high levels of identity and highlighted the close relationship between the members of this group of viruses.

Moving muscles with microchips, mathematics and models: an update on the 'reverse' model.

This paper reviews the current status of our computer model for the management of strabismus and its future direction. Vector analysis was first used in the 1950s for the assessment of strabismus. Robinson's model was the first computer simulation of ocular motility. Using physiological principles and anatomical approximations, Robinson's model sought to predict the strabismic pattern to be expected from a given injury. The Kault/Stark 'reverse' model works in the opposite direction, to first simulate the given strabismic pattern and then advise the surgery required to restore orthophoria. The surgeon is able to 'trial' various operations and compare the expected postoperative results. An automated system is currently being developed to ease the difficulty in measuring the position of the eyes in all nine positions of gaze. This paper includes three illustrative case reports.

Steroid sensitive Acanthamoeba keratitis.

A 44-year-old woman with proven Acanthamoeba keratitis was successfully treated medically with resultant 6/9 vision. During the treatment, repeated attempts to titrate the patient off topical corticosteroids resulted in recurrent flare-up of inflammatory keratitis from which Acanthamoeba could not be recultured. It is suggested that steroid administration during the course of Acanthamoeba keratitis may need to be withdrawn extremely slowly to avoid the recurrence of what appears to be an immunological corneal reaction.