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Background and Purpose: Intraluminal application of pharmacologic agents for acute ureteral dilation may facilitate difficult ureteroscopy. We characterized the in-vivoeffects of intraluminal application of verapamil and theophylline on ureteral peristalsis and diameter in a porcine model.Materials and Methods: Twenty-four female domestic pigs (35–40 kg) were incorporated into the study. We deployed a giant magneto resistive (GMR) sensor and electromagnetic (EMG) electrodes laparoscopically onto the ureteral surface for simultaneous measurement of the mechanical and electrical signals of ureteral peristalsis, respectively. The ureteral-luminal diameter was measured at three levels by digital retrograde pyelography and standardized to a 10-mm laparoscope. The results were calculated as change in peristalsis and ureteral diameter from baseline during the first hour after drug injection. We tested two smooth-muscle relaxants, verapamil (2 mg/kg) and theophylline (70 mg/kg), with saline and dimethylsulfoxide (DMSO; solvent) as controls. Six pigs were studied for each of the four groups. Hydration, anesthesia, and intra-abdominal pressure were standardized. The serum concentrations of the drugs were measured to determine systemic absorption.Results: During the first 10 minutes after intraluminal drug injection, theophylline caused a significant decrease in ureteral peristalsis (6.75 waves/10 minutes) compared with the control group (1.00/10 minutes; P= 0.02). This trend persisted for the next hour. However, there were no changes from baseline in ureteral width. Ureteral peristalsis and dilation remained similar after the saline and DMSO injections. Verapamil increased the diameter of the proximal ureter compared with the controls throughout the hour after drug injection. Fifteen minutes after the drug injection, the change in the ureteral diameter with verapamil was 1.38 mm (4.14F), while the control group showed a change of 0.27 mm (P= 0.03). At 1 hour, the width of the proximal ureter in the verapamil group had increased by 1.72 mm (5.16F), while the control group had changed by 0.55 mm (P= 0.03). There were no statistically significant changes in the widths of the mid or distal ureter. No ureteral dilation was observed in the other groups.Conclusions: In the porcine model, intraluminal application of pharmacologic agents produced independent effects on ureteral dilation and peristalsis. Theophylline inhibited ureteral peristalsis, and verapamil produced acute proximal-ureteral dilation. The ability to alter ureteral diameter or peristaltic activity acutely may facilitate ureteroscopy.

Background and Purpose:The effect of stents on ureteral peristalsis in vivois not entirely clear. We sought to develop a minimally invasive method for its study.Materials and Methods:In female domestic pigs, electrical potentials from the ureter were measured by bipolar steel-wire electromyography electrodes delivered laparoscopically. Mechanical movement was measured by giant magneto resistive sensors mounted on custom-made aluminum strips. After baseline values were obtained, the animals were randomized to receive silicone or polyurethane stents, and ureteral peristalsis was measured for 8 hours acutely and for 4 hours 1 week later.Results:Implantation of the devices took an average of 30 minutes. A consistent correlation was found between laparoscopically observed peristaltic waves and the peristalsis detected by the two measuring devices. The devices themselves did not affect peristalsis. Stent insertion increased peristaltic activity initially but later reduced or stopped it. There was no difference in the effects of the two types of stents.Conclusions:The new technique permits close monitoring of ureteral peristalsis in vivo. Smaller stents appear to have less immediate effect than larger ones, but all type of stents tested eventually caused aperistalsis.

Poliomyelitis is on the verge of eradication as a result of heroic immunization efforts by a consortium including the World Health Organization, Centers for Disease Control and Prevention, United Nations International Children's Emergency Fund and Rotary International. The live vaccine that is the major tool of the program evolves rapidly in vaccine recipients, can spread to their contacts to some extent and can on rare occasions cause poliomyeltis. Some of the difficulties that this implies for the eventual complete cessation of vaccination are proving to be real not theoretical.

ABSTRACTThe isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5'-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigenic characteristics. The significance of the presence in nature of such poliovirus chimeras and the consequences for the current efforts to detect potentially dangerous vaccine-derived poliovirus strains are discussed in the context of the global polio eradication initiative.

ABSTRACTCHAT and Cox type 1 live-attenuated poliovirus strains were developed in the 1950s to be used as vaccines for humans. This paper describes their characterization with respect to virulence, sensitivity for growth at high temperatures, and complete nucleotide and amino acid sequences. The results are compared to those for their common parental wild virus, the Mahoney strain, and to those for two other poliovirus strains derived from Mahoney, the Sabin 1 vaccine strain and the mouse-adapted LS-a virus. Analysis of four isolates from cases of vaccine-associated paralytic poliomyelitis related to the CHAT vaccine revealed genetic and phenotypic properties of the CHAT strain following replication in the human gut. CHAT-VAPP strain 134 contained a genome highly evolved from that of CHAT (1.1% nucleotide differences), suggesting long-term circulation of a vaccine-derived strain in the human population. The molecular mechanisms of attenuation and evolution of poliovirus in humans are discussed in the context of the global polio eradication initiative.

A clear association was demonstrated between the use of USOL-D-bac type 3 poliovirus live-attenuated vaccine and the 1968 poliomyelitis epidemic in Poland. The epidemic followed small-scale trials with Sabin and USOL-D-bac type 3 vaccine strains carried out in seven countries including Poland. Factors that might have contributed to the genesis and development of the epidemic were the pattern of virus excretion from vaccinees, mutations found in viruses from the epidemic, and the particular vaccination policies in Poland during the previous years. These findings may provide essential insights into the strategies for stopping polio immunisation once wild poliovirus has been eradicated.

Polioviruses are single-stranded RNA viruses with an unusually long noncoding region (NCR) at the 5′ end predicted to have an elaborate secondary structure made up of six domains. Mutations in domain V of the poliovirus 5′NCR that disrupt secondary structure are responsible for attenuation of the virus and a temperature-sensitive (ts) phenotype in vitro. In addition to direct back mutation or compensatory second site mutation in the 5′NCR as previously documented, the ts phenotype was found to be compensated for in monkey kidney cells in vitroby a coding change in the protease 2A. These coding changes were found throughout the protease with no obvious pattern or trend. They were not all found to be equivalent and limited in ability to compensate for the severest domain V disruption. The compensatory effect of the 2A changes was found to be cell specific, having no effect on monkey neurovirulence and in a mouse cell line but a significant effect in two monkey cell lines and a human epithelial line.

ABSTRACTA 20-year-old female hypogammaglobulinemic patient received monotypic Sabin 3 vaccine in 1962. The patient excreted type 3 poliovirus for a period of 637 days without developing any symptoms of poliomyelitis, after which excretion appeared to have ceased spontaneously. The evolution of Sabin 3 throughout the entire period of virus excretion was studied by characterization of seven sequential isolates from the patient. The isolates were analyzed in terms of their antigenic properties, virulence, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 3 vaccine. The isolates followed a main lineage of evolution with a rate of nucleotide substitution that was very similar to that estimated for wild-type poliovirus during person-to-person transmission. There was a delay in the appearance of antigenic variants compared to sequential type 3 isolates from healthy vaccines, which could be one of the possible explanations for the long-term excretion of virus from the patient. The distribution of mutations in the isolates identified regions of the virus possibly involved in adaptation for growth in the human gut and virus persistence. None of the isolates showed a full reversion of the attenuated and temperature-sensitive phenotypes of Sabin 3. Information of this sort will help in the assessment of the risk of spread of virulent polioviruses from long-term excretors and in the design of therapies to stop long-term excretion. This will make an important contribution to the decision-making process on when to stop vaccination once wild poliovirus has been eradicated.

ABSTRACTBinding of yellow fever virus wild-type strains Asibi and French viscerotropic virus and vaccine strains 17D and FNV to monkey brain and monkey liver cell membrane receptor preparations (MRPs) was investigated. Only FNV bound to monkey brain MRPs, while French viscerotropic virus, Asibi, and FNV all bound to monkey liver MRPs. Four monkey brain and two mouse brain MRP escape (MRPR) variants of FNV were selected at pH 7.6 and 6.0. Three monkey brain MRPRvariants selected at pH 7.6 each had only one amino acid substitution in the envelope (E) protein in domain II (E-237, E-260, or E274) and were significantly attenuated in mice following intracerebral inoculation. Two of the variants were tested in monkeys and retained parental neurotropism following intracerebral inoculation at the dose tested. We speculate that this region of domain II is involved in binding of FNV E protein to monkey brain and is, in part, responsible for the enhanced neurotropism of FNV for monkeys. A monkey brain MRPRvariant selected at pH 6.0 and two mouse brain MRPRvariants selected at pH 7.6 were less attenuated in mice, and each had an amino acid substitution in the transmembrane region of the E protein (E-457 or E-458).

Six passages of the mosquito-borne flavivirus yellow fever (YF) wild-type strain Asibi in HeLa cells attenuated the virus for monkeys and newborn mice and resulted in loss of mosquito competence. Attenuation after the passage in HeLa cells was not unique to YF virus strain Asibi as demonstrated by the HeLa passage attenuation of wild-type YF virus strain French viscerotropic virus and YF vaccine virus 17D-204 for newborn mice. In contrast, wild-type strain Dakar 1279 and the French neurotropic vaccine virus remained virulent for newborn mice after six passages in HeLa cells. Thus not all strains of YF virus can be attenuated by passage in HeLa cells. Attenuation of YF virus strains Asibi and French viscerotropic virus was accompanied by alterations in the antigenic and biological properties of the viruses, including changes to envelope protein epitopes. Attenuation for newborn mice was coincidental with the acquisition by the HeLa-passaged viruses of the vaccine-specific envelope protein epitope recognized by monoclonal antibody H5. This suggests that this conformational change may play a role in the attenuation process. Wild-type Dakar 1279, which remained virulent for newborn mice after passage in HeLa cells, retained its wild-type antigenic character. The genome of Asibi HeLa p6 virus differed from wild-type Asibi virus by 29 nucleotides that encoded 10 amino acid substitutions: 5 in the envelope protein, 1 in NS2A, 3 in NS4B, and 1 in NS5. The substitution at NS4B-95 is seen in three different attenuation processes of wild-type YF virus, leading us to speculate that it is involved in the attenuation of virulence of wild-type strain Asibi.

The three‐dimensional structure of the Sabin strain of type 3 poliovirus has been determined at 2.4 A resolution. Significant structural differences with the Mahoney strain of type 1 poliovirus are confined to loops and terminal extensions of the capsid proteins, occur in all of the major antigenic sites of the virion and typically involve insertions, deletions or the replacement of prolines. Several newly identified components of the structure participate in assembly‐dependent interactions which are relevant to the biologically important processes of viral assembly and uncoating. These include two sites of lipid substitution, two putative nucleotides and a beta sheet formed by the N‐termini of capsid proteins VP4 and VP1. The structure provides an explanation for the temperature sensitive phenotype of the P3/Sabin strain. Amino acids that regulate temperature sensitivity in type 3 poliovirus are located in the interfaces between promoters, in the binding site for a lipid substituent and in an assembly‐dependent extended beta sheet that stabilizes the association of pentamers. Several lines of evidence indicate that these structural components also control conformational transitions at various stages of the viral life cycle.

To determine the effects of κ-opioid receptor agonists on phosphoinositide metabolism in rat renal cortex, tissue slices labelled with [ 3 H]inositol were stimulated with norepinephrine or carbachol alone or in combination with the κ-opioid receptor agonists, ethylketocyclazocine, trans-3,4-dichloro- N -methyl- N -[2-(pyrrolindinyl)-cyclohexyl)-benzeneacetamide (U50,488) and nalorphine. Both norepinephrine and carbachol stimulated phosphoinositide hydrolysis (measured in a LiCl buffer) concentration- and time-dependently. The EC 50 and maximal stimulation of phosphoinositide hydrolysis for norepinephrine and carbachol were approximately 3 μM and 0.15 dpm released/dpm incorporated, respectively. Concentrations up to 1 mM of ethylketocyclazocine, U50,488 or nalorphine alone did not affect phosphoinositide hydrolysis. However, ethylketocyclazocine and U50,488 decreased 10μM norepinephrine-stimulated phosphoinositide hydrolysis concentration-dependently, each with an approximate IC 50 of 30 μM. In contrast, nalorphine had no effect on norepinephrine-stimulated phosphoinositide hydrolysis. In addition, concentrations of up to 1 mM ethyketocyclazocine or U50,488 did not alter carbachol-stimulated phosphoinositide hydrolysis. The inhibitory effect of U50,488 and ethylketocyclazocine on norepinephrine-stimulated phosphoinositide hydrolysis was blocked by the selective κ-opioid receptor antagonists, nor-binaltorphimine. These results indicate that κ 1 - opioid receptor stimulation may effect phosphoinositide metabolism in rat renal cortex by modulating the subcellular effects of renal α 1 - adrenoceptor activation.

Mouse liver perfusion studies in situ revealed that the cholinesterase inhibitor chlorpyrifos oxon produced by the liver from the phosphorothioate pesticide chlorpyrifos was quickly detoxified within the liver, thereby preventing it's exit from the liver in the effluent. In contrast, when the pesticide parathion was perfused as a substrate a substantial amount of the toxic metabolite paraoxon was found in exiting perfusate. Pesticide concentrations (5-15 microM) used in the perfusion studies in situ were similar to their hepatic portal blood concentrations in vivo (2.32-12.95 microM) after i.p. administration of lethal or near lethal doses. Moreover, the half-life for elimination of paraoxon by mouse blood in vitro was 8.6 min, a rate sufficiently low to allow passage of paraoxon to extrahepatic target tissues from liver in vivo. These results suggest that in the mouse, the acute toxicity of chlorpyrifos is mediated by extrahepatic production of oxon, whereas that of parathion is likely mediated by both hepatic and extrahepatic activation.

The envelope (E) protein genes of the wild-type yellow fever (YF) virus French viscerotropic virus and its' vaccine derivative, French neurotropic virus (FNV), were compared and found to differ by 13 nucleotides that coded for 8 amino acid substitutions. Comparison of the E proteins of FNV and 17D, the vaccine strain derived from wild-type strain Asibi, showed that there was no common nucleotide change or amino acid substitution between these two vaccine strains. However, changes are clustered around amino acid positions 52-56 and may represent the common vaccine epitope shared by 17D and FNV vaccine viruses. The molecular basis of any difference in neurotropism and viscerotropism of YF virus, attributable to the E protein, remains unclear. Copyright 1993, 1999 Academic Press

As part of a study into the molecular basis of attenuation and reversion to neurovirulence in the Sabin poliovirus vaccines, we have determined the complete nucleotide sequence of a cloned DNA copy of the genome of P3/Leon/37, the neurovirulent progenitor of the type 3 Sabin vaccine strain, P3/Leon 12a1b. Comparison of the sequence with that which we previously obtained for the vaccine strain [Stanway, G., Cann, A. J., Hauptmann, R., Hughes, P., Clarke, L. D., Mountford, R. C., Minor, P. D., Schild, G. C. & Almond, J. W. (1983) Nucleic Acids Res. 11, 5629-5643] indicates that attenuation has been brought about by a maximum of 10 point mutations, at least 5 of which are likely to be of minor significance. Predicted amino acid sequences of all the known virus-encoded proteins show that amino acid substitutions have occurred at only three positions. Two of these are in structural proteins (i.e., Ser----Phe in VP3 and Lys----Arg in VP1), and the third, Thr----Ala, is in the nonstructural protein P2-3b. The distribution and nature of nucleotide and amino acid sequence differences suggest that a single base substitution may be responsible for the attenuated phenotype of the vaccine strain.

A study of the biochemistry of type 3 poliovirus strains which involves the examination of the virus-coded polypeptides in infected cells and the preparation of oligonucleotide maps is reported. The polypeptide patterns were shown to be a relatively stable property of virus strains and distinguished Sabin vaccine strains from wild strains of poliovirus type 3. This approach may be of value in deciding the origin (vaccine or nonvaccine) of field isolates of poliovirus. Oligonucleotide maps were found to be sensitive indicators of differences among strains and appear to form a basis for determining genetic relationships among strains. The nucleotide maps of two viruses isolated from human cases of paralytic poliomyelitis temporally associated with the administration of attenuated vaccine suggested a vaccine origin for the strain. In one case the nucleotide map was indistinguishable from that of the vaccine strain.

The transmission of viral infections through the use of products derived from blood has emphasised the need for adequate validation of the production process, testing of materials used in production and quality control tests on the final product. Since the late 1980s, as part of its batch release procedures, NIBSC has tested for markers of viral infectivity plasma pools used in production of blood products used in the UK. As a result of testing over 9,000 pools, NIBSC has identified 9 pools contaminated with HBsAg and 2 pools containing antibodies to HIV‐1. Since routine screening of plasma pools for anti‐HCV was introduced in 1993, 8 pools out of the 4,000 tested have been found to contain antibodies to HCV. In addition, the release of 12 batches of blood products was withheld and it is known that further batches of material produced from the positive pools were not submitted for batch release. Studies involving assays of dilutions of known positive plasma samples indicated that there is considerable variation in the endpoint dilutions of antigen or antibody detected by test kits from different manufacturers. The selection and validation of the kits used in such testing is therefore important. The usefulness of standardised low‐level external controls in assays of plasma pools for markers of viral infection is discussed.

We have determined a major antigenic site for virus neutralization on the capsid protein VP1 of poliovirus type 3. Antigenic mutant viruses selected for resistance to individual monoclonal antibodies had point mutations concentrated in a region 277–294 bases downstream from the start of the region of viral RNA coding for VP1. These findings provide the basis for an improved understanding of the molecular basis of virus neutralization.