Baculovirus phosphoprotein pp31 is associated with virogenic stroma. The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle.


Attenuation of Sindbis virus neurovirulence by using defined mutations in nontranslated regions of the genome RNA. We examined a panel of Sindbis virus mutants containing defined mutations in the 5' nontranslated region of the genome RNA, in the 3' nontranslated region, or in both for their growth in cultured cells and virulence in newborn mice. In cultured cells, these viruses all had defects in RNA synthesis and displayed a wide range of growth rates. The growth properties of the mutants were often very different in mouse cells from those in chicken cells or in mosquito cells. We hypothesize that host factors, presumably proteins, interact with these nontranslated regions to promote viral replication and that the mammalian protein and the chicken or mosquito protein are sufficiently divergent that alterations in the viral RNA sequence can affect the interactions with these different host proteins in different ways. Some of the mutants were temperature sensitive for plaque formation, whereas one mutant was slightly cold sensitive in its growth in chicken cells. Upon inoculation into mice, viruses that grew well in cultured mouse cells retained their virulence, but mice that succumbed usually had extended survival times. One virulent mutant that grew slightly less well in cultured mouse cells than did the parental virus produced eight times as much virus in mouse brain following intracerebral inoculation, suggesting that changes in these regulatory regions may have tissue-specific as well as host-specific effects. Viruses that were severely crippled in their growth in mouse cells in culture were usually, but not always, attenuated in their virulence. In particular, temperature sensitivity was correlated with attenuation. The effect of two mutations was found to be cumulative, and double mutants that contained mutations in both the 5' and 3' nontranslated regions were more attenuated than was either single mutant. Three of four double mutants tested were severely crippled for virus production in cultured cells and were avirulent for mice, even when inoculated intracerebrally.


Mutational inactivation of an inhibitory sequence in human immunodeficiency virus type 1 results in Rev-independent gag expression. We have characterized an inhibitory RNA element in the human immunodeficiency virus type 1 (HIV-1) gag coding sequence that prevents gag expression. The inhibition exerted by this element could be overcome by the presence of the Rev-responsive element in cis and of Rev protein in trans. To understand the mechanism of function, we inactivated the inhibitory element by mutagenesis while maintaining an intact gag coding region. A constitutive high level of Rev-independent gag expression was achieved only after the introduction of 28 point mutations over a large region of 270 nucleotides within the gag coding region. To our knowledge, this is the first demonstration of inactivation of a negative RNA element within a coding region without alteration of the expressed protein. Elimination of the inhibitory element in the p17gag region, named INS-1, offered the opportunity to detect a second inhibitory element in the gag-pol region. The presence of either INS element is sufficient to inhibit gag expression, demonstrating that multiple INS elements acting independently can inhibit HIV RNA expression. Expression of gag from Rous sarcoma virus, a retrovirus that does not require Rev-like regulatory proteins, revealed that the Rous sarcoma virus p19gag region does not contain inhibitory elements. These results demonstrate the presence of a strong inhibitory element acting at the level of mRNA and provide a general method for the removal of such elements from mRNA coding regions. The inhibitory element functions in the absence of any HIV-1 proteins, suggesting that cellular factors are responsible for this inhibition.


Mutational analysis of human T-cell leukemia virus type I Tax: regions necessary for function determined with 47 mutant proteins. We have made 47 mutations that span the length of the human T-cell leukemia virus type I (HTLV-I) Tax open reading frame. Of the 47 mutations, 38 were substitutions of single amino acids, 5 were missense changes in two or more amino acids, and 4 were deletions. A subset of these mutations includes individual changes of all 26 naturally occurring serines to alanines. By assaying each mutant protein separately on the HTLV-I long terminal repeat (LTR) and the human immunodeficiency virus type 1 (HIV-1) LTR in parallel, we were able to identify regions of Tax selectively necessary for each promoter. A small region in the carboxyl terminus, amino acids 315 to 325, was found to be selectively important for activation of the HTLV-I LTR. Three changes at serine 113, serine 160, and serine 258 were found to specifically affect function on the HIV-1 LTR. Surprisingly, we found that the great preponderance of missense changes (32 of 42) in Tax did not affect function.


Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. CD4 is an integral membrane glycoprotein which is known as the human immunodeficiency virus (HIV) receptor for infection of human cells. The protein is synthesized in the endoplasmic reticulum (ER) and subsequently transported to the cell surface via the Golgi complex. HIV infection of CD4+ cells leads to downmodulation of cell surface CD4, due at least in part to the formation of stable intracellular complexes between CD4 and the HIV type 1 (HIV-1) Env precursor polyprotein gp160. This process "traps" both proteins in the ER, leading to reduced surface expression of CD4 and reduced processing of gp160 to gp120 and gp41. We have recently demonstrated that the presence of the HIV-1-encoded integral membrane protein Vpu can reduce the formation of Env-CD4 complexes, resulting in increased gp160 processing and decreased CD4 stability. We have studied the effect of Vpu on CD4 stability and found that Vpu induces rapid degradation of CD4, reducing the half-life of CD4 from 6 h to 12 min. By using a CD4-binding mutant of gp160, we were able to show that this Vpu-induced degradation of CD4 requires retention of CD4 in the ER, which is normally accomplished through its binding to gp160. The involvement of gp160 in the induction of CD4 degradation is restricted to its function as a CD4 trap, since, in the absence of Env, an ER retention mutant of CD4, as well as wild-type CD4 in cultures treated with brefeldin A, a drug that blocks transport of proteins from the ER, is degraded in the presence of Vpu.


U3 long terminal repeat-mediated induction of intracellular immunity by a murine retrovirus: a novel model of latency for retroviruses. BL/VL3 radiation leukemia virus (RadLV) is a thymotropic, highly leukemogenic murine leukemia virus (MuLV) which is unable to replicate in vitro in mouse fibroblasts. We have previously reported that the U3 long terminal repeat region of its genome is responsible for this block (E. Rassart, Y. Paquette, and P. Jolicoeur, J. Virol. 62:3840-3848, 1988). By using hybrids of permissive and resistant cells infected with BL/VL3 RadLV or fibrotropic MuLV, we found that the resistant phenotype was dominant. Investigation to determine at which step of the virus cycle the block operates revealed that integration, transcription, and translation of the BL/VL3 viral genome occurred at normal levels in nonpermissive cells. The BL/VL3 RadLV Pr65gag proteins made in nonpermissive cells were also myristylated and located at the membrane, and the levels of their cleaved products were similar to those of fibrotropic MuLV. However, processing of BL/VL3 RadLV Pr85env was impaired in nonpermissive cells. Virions were not released into the culture medium of nonpermissive cells, as measured by reverse transcriptase activity and by content in p30 or gp70 protein and as documented by lower levels of budding particles seen by electron microscopy. These results indicate that BL/VL3 RadLV replication is blocked at a late stage of the virus cycle, i.e., at virion assembly. Interestingly, these BL/VL3 RadLV-infected nonpermissive fibroblasts were resistant to superinfection by fibrotropic Moloney MuLV, and this resistance also occurred at a late step of the Moloney virus cycle. Since this block is dominant, it appears that the U3 long terminal repeat region of the BL/VL3 viral genome has the ability to induce a cellular suppressor factor(s), thus bringing intracellular immunity against itself and against other ecotropic MuLVs.


Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope. Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.


Mutational analysis of the octapeptide sequence motif at the NS1-NS2A cleavage junction of dengue type 4 virus. We have previously shown that proper processing of dengue type 4 virus NS1 from the NS1-NS2A region of the viral polyprotein requires a hydrophobic N-terminal signal and the downstream NS2A. Results from deletion analysis indicate that a minimum length of eight amino acids at the C terminus of NS1 is required for cleavage at the NS1-NS2A junction. Comparison of this eight-amino-acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala. Site-directed mutagenesis was performed to construct mutants of NS1-NS2A that contained a single amino acid substitution at different positions of the consensus cleavage sequence or at the immediate downstream position. Three to eight different substitutions were made at each position. A total of 50 NS1-NS2A mutants were analyzed for their cleavage efficiency relative to that of the wild-type dengue type 4 virus sequence. As predicted, nearly all substitutions at positions P1, P3, P5, P7, and P8, occupied by conserved amino acids, yielded low levels of cleavage, with the exception that Pro or Ala substituting for Ser (P5) was tolerated. Substitutions of an amino acid at the remaining positions occupied by nonconserved amino acids generally yielded high levels of cleavage. However, some substitutions at nonconserved positions were not tolerated. For example, substitution of Gly or Glu for Gln (P4) and substitution of Val or Glu for Lys (P6) each yielded a low level of cleavage. Overall, these data support the proposed cleavage sequence motif deduced by comparison of sequences among the flaviviruses. This study also showed that in addition to the eight-amino-acid sequence, the amino acid immediately following the NS1-NS2A cleavage site plays a role in cleavage.


Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site. The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins induce cytoplasmic expression of incompletely spliced viral mRNAs by binding to these mRNAs in the nucleus. Each protein binds a specific cis-acting element in its target RNAs. Both proteins also associated with nucleoli, but the significance of this association is uncertain because mutations that inactivate nucleolar localization signals in Rev or Rex also prevent RNA binding. Here we demonstrate that Rev and Rex can function when tethered to a heterologous RNA binding site by a bacteriophage protein. Under these conditions, cytoplasmic accumulation of unspliced RNA occurs without the viral response elements, mutations in the RNA binding domain of Rev do not inhibit function, and nucleolar localization can be shown to be unnecessary for the biological response.


Expression and characterization of the trans-activating protein Tax of human T-cell leukemia virus type I in Saccharomyces cerevisiae. The trans-activator protein Tax of human T-cell leukemia virus type I (HTLV-I) stimulates transcription of the viral genome from the long terminal repeat. With a reporter HIS4TATA::lacZ fusion gene, the transcriptional activity of the Tax-responsive element in the long terminal repeat was tested in Saccharomyces cerevisiae. We found that fragments containing the 21-bp repeat of the HTLV-I enhancer stimulate synthesis of beta-galactosidase activity 15- to 20-fold. To test the ability of the Tax protein to trans activate the HTLV-I enhancer in yeast cells, the pX region of HTLV-I, encoding the Tax protein, was cloned under the control of the yeast GAL1 promoter. The expressed Tax protein is localized in the nucleus and associated with the yeast nuclear matrix fraction. In yeast cells that contained the integrated tax gene, two- to sixfold stimulation of expression from the HTLV-I enhancer was detected at the early stages of tax induction. This in vivo reconstitution system provides a new approach for examining the host factor(s), the signal transduction mechanism(s), and the role of nuclear architecture involved in Tax-mediated trans activation.


The requirements for viral entry differ from those for virally induced syncytium formation in NIH 3T3/DTras cells exposed to Moloney murine leukemia virus. Moloney murine leukemia virus (Mo-MuLV) has the unique ability to infect different cells via either a low-pH-dependent or a pH-independent entry pathway. Only the pH-independent mechanism of Mo-MuLV entry has been associated with Mo-MuLV-induced syncytium formation. We have now identified a transformed cell line (NIH 3T3/DTras) which efficiently forms syncytia when exposed to Mo-MuLV, yet is low pH dependent for Mo-MuLV entry. Treatment of NIH 3T3/DTras cells with chloroquine, an agent which raises endosomal pH, blocks Mo-MuLV entry, but not Mo-MuLV-induced syncytium formation. This demonstrates that fusion which accompanies viral entry and fusion which is responsible for syncytium formation occur as independent processes in these cells. In addition, we determined that neither inherent differences in the Mo-MuLV receptor nor reduced affinity for Mo-MuLV gp70 can account for resistance of NIH 3T3 cells to Mo-MuLV-induced syncytium formation.


Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus. Brome mosaic virus is a positive-strand RNA virus whose RNA replication requires viral protein 1a, which has putative helicase and capping functions, and 2a, which has putative polymerase function. Since domains of related sequence are conserved in a wide range of plus-strand RNA viruses, analysis of 1a and 2a function should have applicability to many other viruses. We have recently demonstrated that 1a and 2a form a complex in vivo and in vitro. Using immune coprecipitation and mutant polypeptides made in reticulocyte lysates, we have now mapped both the 1a and 2a domains necessary for complex formation. The sequences needed to bind 2a map to the carboxy-terminal helicase-like domain of 1a. Truncated polypeptides containing this domain were able to bind to 2a, while several small insertions in the helicase-like domain disrupted binding. The sequence required for binding 1a lies within a 115-residue subset of the 2a N-terminal segment preceding the polymerase-like domain. Truncations or fusion polypeptides containing this segment can bind 1a. We also determined that highly purified 2a protein made in insect cells can form a complex with highly purified 1a helicase-like domain made in Escherichia coli, suggesting that no other factor is required to mediate 1a-2a interaction. Previous genetic analyses of 1a and 2a are consistent with this mapping and show that the newly defined 1a and 2a binding regions are required for RNA synthesis. The locations of these interacting regions are discussed with regard to models of viral replication and the evolution of positive-strand RNA virus genomes.


Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein. Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated fusion process of the alphavirus Semliki Forest virus (SFV). The spike protein of SFV is composed of three copies of the protein heterodimer E2E1. This structure is resistant to solubilization in mild detergents such as Nonidet P-40 (NP40). We have recently shown that the spike structure is reorganized during virus entry into acidic endosomes (J. M. Wahlberg and H. Garoff, J. Cell Biol. 116:339-348, 1992). The original NP40-resistant heterodimer is dissociated, and the E1 subunits form new NP40-resistant protein oligomers. Here, we show that the new oligomer is represented by an E1 trimer. From studies that use an in vitro assay for fusion of SFV with liposomes, we show that the E1 trimer is efficiently expressed during virus-mediated membrane fusion. Time course studies show that both E1 trimer formation and fusion are fast processes, occurring in seconds. It was also possible to inhibit virus binding and fusion with a monoclonal antibody directed toward the trimeric E1. These results give support for a model in which the E1 trimeric structure is involved in the SFV-mediated fusion reaction.


Molecular profile of human immunodeficiency virus type 1 infection in symptomless patients and in patients with AIDS. Recent molecular evidence indicates that active human immunodeficiency virus type 1 (HIV-1) infection is detectable in both symptomless and symptomatic infected patients. For this main reason, it has been pointed out that precise quantitative analysis of viral activity in vivo is necessary, firstly, for the pathogenetic investigation of the steps relevant to infection progression and, secondly, for better clinical management of HIV-1-infected patients. In this study, the presence of HIV-1 genomic RNA in plasma samples, specific HIV-1 transcripts in peripheral blood mononuclear cells, and proviral DNA sequences were assayed for 33 HIV-1-infected patients (including symptomless and symptomatic subjects) by using a competitive polymerase chain reaction method that allows quantitation of the RNA/DNA target sequences. The quantitative results obtained confirm that transcription of HIV-1 structural genes and complete viral replication occur in all the HIV-1-infected patients independently of the clinical stage. However, although sharp individual differences were detected, a high degree of correlation of the molecular parameters studied with both disease progression and a decrease in the number of CD4+ T lymphocytes was documented. Interestingly, despite the increasing viremia level associated with infection progression, the mean transcriptional activity of individual infected cells was found to be only moderately greater in AIDS patients than in asymptomatic infected subjects. In addition, it was noted that quantitation of HIV-1 genomic RNA in plasma samples and quantitation of specific HIV-1 transcripts in peripheral blood mononuclear cells appear to be more reliable and sensitive markers of viral activity than quantitative analysis of proviral HIV-1 sequences in peripheral lymphocytes.


Promoter influence on baculovirus-mediated gene expression in permissive and nonpermissive insect cell lines. The activities of viral and insect promoters were examined in a range of insect cell lines permissive and nonpermissive for the replication of the baculovirus Autographa californica nuclear polyhedrosis virus. Recombinant baculoviruses were constructed to place the bacterial chloramphenicol acetyltransferase gene under the control of promoters strongly active in the early, late, or very late stages of virus replication. In fully permissive cells, expression from a very late promoter was 2- to 3-fold higher than expression from a late promoter and 10- to 20-fold higher than expression from an early promoter or from a virus-borne insect promoter. In cell lines that do not support the efficient production of viral progeny, late-promoter-driven expression was similar to or surpassed very late promoter-driven expression. In nonpermissive insect cell lines, expression driven by an insect promoter derived from Drosophila melanogaster was higher than expression from the three viral promoters and was especially high in the Drosophila cell line tested. Surprisingly, late-promoter-driven expression, which is dependent on DNA replication, was higher than early-promoter-driven expression in three of four nonpermissive lines. In contrast, very late promoter-driven expression was quite limited in nonpermissive cell lines. The results indicate that the promoter used to drive foreign-gene expression strongly influences the range of insect cells which can efficiently support the production of the foreign protein during infection with recombinant baculoviruses.


Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells. The human immunodeficiency virus type 1 (HIV-1) integrase enzyme exhibits significant amino acid sequence conservation with integrase proteins of other retroviruses. We introduced specific amino acid substitutions at a number of the conserved residue positions of recombinant HIV-1 integrase. Some of these substitutions resulted in proteins which were not able to be purified in the same manner as the wild-type enzyme, and these were not studied further. The remaining mutant enzymes were assessed for their abilities to perform functions characteristic of the integrase protein. These included specific removal of the terminal dinucleotides from oligonucleotide substrates representative of the viral U5-long terminal repeat, nonspecific cleavage of oligonucleotide substrates, and mediation of the strand transfer (integration) reaction. Substitution at position 43, within the protein's zinc finger motif region, resulted in an enzyme with reduced specificity for cleavage of the terminal dinucleotide. In addition, a double substitution of aspartic acid and glutamine for valine and glutamic acid, respectively, at positions 151 and 152 within the D,D(35)E motif region rendered the integrase protein inactive for all of its functions. The introduction of this double substitution into an infectious HIV-1 provirus yielded a mutant virus that was incapable of productively infecting human T-lymphoid cells in culture.


Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate-early 1 transcriptional regulatory protein. Autographa californica multicapsid nuclear polyhedrosis virus-infected insect cells express a viral immediate-early transcriptional regulatory protein, IE1, that has been shown by transient-expression assays to stimulate the expression of certain baculovirus delayed-early (DE) promoters and to inhibit the expression of other immediate-early (IE) genes. It is believed that certain DE promoters are activated, in part, by direct interactions between IE1 and enhancer elements located in regions adjacent to these genes. We have used transient cotransfection and DNA-binding assays to examine the function of mutant forms of IE1. Our results indirectly show that IE1 has at least two separable domains that are essential for its role in the modulation of baculovirus gene expression. A domain rich in acidic residues and essential for transactivation is located within the N-terminal 145 amino acids of the polypeptide. A second domain, located in the C-terminal 437 amino acids of IE1, is required for inhibitory and DNA-binding activities. Several nontransactivating IE1 mutants trans-dominantly interfered with wild-type IE1 transactivation of enhancer-linked DE genes. trans-dominant interference was expressed only by IE1 mutants that retained the N-terminal putative acidic activation domain, suggesting that this region may be involved in associations with a factor(s) essential for activation of enhancer-linked genes.


Pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of CD4+ T cells. In previous studies, it was observed that children immunized with a formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) developed severe pulmonary disease with greater frequency during subsequent natural RSV infection than did controls. During earlier efforts to develop an animal model of this phenomenon, enhanced pulmonary histopathology was observed after intranasal RSV challenge of FI-RSV-immunized cotton rats. Progress in understanding the immunologic basis for these observations has been hampered by the lack of reagents useful in manipulating the immune response of the cotton rat. This problem prompted us to reinvestigate the characteristics of immunity to RSV in the mouse. In the present studies, extensive pulmonary histopathology was observed in FI-RSV-immunized or RSV-infected BALB/c mice upon RSV challenge, and studies to determine the relative contributions of CD4+ or CD8+ T cells to this process were undertaken. Mice previously immunized with FI-RSV or infected with RSV were depleted of CD4+, CD8+, or both T-cell subsets immediately prior to RSV challenge, and the magnitude of inflammatory cell infiltration around bronchioles and pulmonary blood vessels and into alveolar spaces was quantified. The magnitude of infiltration at each anatomic site in previously FI-RSV-immunized or RSV-infected, nondepleted animals was similar, indicating that this is not a relevant model for enhanced disease. However, the effect of T-cell subset depletion on pulmonary histopathology following RSV challenge was very different between the two groups. Depletion of CD4+ T cells completely abrogated pulmonary histopathology in FI-RSV-immunized mice, whereas it had a much smaller effect on mice previously infected with RSV. FI-RSV-immunized or RSV-infected animals depleted of CD8+ T cells had only a modest reduction of pulmonary histopathology. In addition, RSV infection induced high levels of major histocompatibility complex class I-restricted cytotoxic T-cell activity, whereas FI-RSV immunization induced a low level. These data indicate that immunization with FI-RSV induces a cellular immune response different from that induced by RSV infection, which likely played a role in enhanced disease observed in infants and children.


Human immunodeficiency virus type 1 Rev activation can be achieved without Rev-responsive element RNA if Rev is directed to the target as a Rev/MS2 fusion protein which tethers the MS2 operator RNA. The posttranscriptional trans activation of unspliced or partially spliced human immunodeficiency virus RNAs by the Rev regulatory protein is crucial for virus replication and is dependent on sequence-specific RNA binding by Rev. The cognate RNA target of Rev is contained within a highly structured, 244-nucleotide Rev-responsive element (RRE) RNA in the viral env gene. Here, we show that specific interaction with the RRE is not an absolute requirement for Rev function. When the RRE is replaced by a heterologous MS2 phage operator sequence, Rev will facilitate the cytoplasmic expression of human immunodeficiency virus mRNAs containing this sequence if directed to the MS2 operator via the RNA binding motif of the MS2 phage coat protein (MS-C) as a Rev/MS-C fusion protein. Rev/MS-C efficiently activated both RRE and MS2 targets. A mutation in the MS2 operator that abolished the coat protein binding in vitro rendered the mutant RNA nonresponsive to the fusion protein in vivo. Notwithstanding that Rev can be tethered to the viral RNAs via another RNA binding motif, the structural integrity of the N terminus of Rev was still required for optimal trans activation.


An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. We isolated and molecularly cloned a human immunodeficiency virus type 1 (HIV-1) strain (89.6) which is unusual because it is both macrophage-tropic and extremely cytopathic in lymphocytes. Moreover, this is the first well-characterized infectious molecularly cloned macrophage-tropic HIV-1 strain derived from peripheral blood. HIV-1 89.6 differs markedly from other macrophage-tropic isolates within the envelope V3 region, which is important in determining cell tropism and cytopathicity. HIV-1 89.6 may thus represent a transitional isolate between noncytopathic macrophage-tropic viruses and cytopathic lymphocyte-tropic viruses.


TAR-independent replication of human immunodeficiency virus type 1 in glial cells. The molecular mechanisms involved in the replication of human immunodeficiency virus type 1 (HIV-1) may differ in various cell types and with various exogenous stimuli. Astrocytic glial cells, which can support HIV-1 replication in cell cultures and may be infected in vivo, are demonstrated to provide a cellular milieu in which TAR mutant HIV-1 viruses may replicate. Using transfections of various TAR mutant HIV-1 proviral constructs, we demonstrate TAR-independent replication in unstimulated astrocytic cells. We further demonstrate, using viral constructs with mutations in the tat gene and in the nuclear factor kappa B (NF-kappa B)-binding sites (enhancer) of the HIV-1 long terminal repeat, that TAR-independent HIV-1 replication in astrocytic cells requires both intact NF-kappa B moiety-binding motifs in the HIV-1 long terminal repeat and Tat expression. We measured HIV-1 p24 antigen production, syncytium formation, and levels and patterns of viral RNA expression by Northern (RNA) blotting to characterize TAR-independent HIV-1 expression in astrocytic glial cells. This alternative regulatory pathway of TAR-independent, Tat-responsive viral production may be important in certain cell types for therapies which seek to perturb Tat-TAR binding as a strategy to interrupt the viral lytic cycle.


Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody. The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is thought to induce potent neutralizing antibodies which are generally defined as type specific and reactive with individual viral isolates. In contrast, the CD4-binding domain is thought to induce neutralizing antibodies that are group specific and capable of neutralizing all isolates of HIV-1. However, in this study, we used a panel of human monoclonal antibodies to these regions of gp120 which displays specificities and neutralizing activities that challenge these tenets. In particular, we used a human monoclonal antibody to the V3 domain with exceptionally potent and broad neutralizing activity against many diverse HIV-1 isolates. The anti-CD4-binding domain antibodies, on the other hand, showed a more restricted pattern of activity.


Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5. Processing of dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5 could be mediated by the catalytically active NS3 protease domain and NS2B in trans at the dibasic sites NS3-NS4A and NS4B-NS5. Subcellular localization of the unprocessed precursor NS3-NS4A-NS4B-NS5 showed that it was confined to a distinct subcellular organelle in the cytoplasm, which was distinct from the distribution of the mature NS5.


Specific binding of the human T-cell leukemia virus type I Rex protein to a short RNA sequence located within the Rex-response element. Expression of the structural proteins of human T-cell leukemia virus type I is dependent upon the interaction of the viral Rex trans activator with its highly structured cis-acting RNA target sequence, the 254-nucleotide Rex-response element. Nucleotides critical for Rex binding in vitro have been mapped by modification interference analysis to a discrete 12-nucleotide RNA sequence that is predicted to form a stem-bulge-stem structure. This minimal RNA binding site was sufficient to mediate specific Rex binding in vitro when analyzed in the context of a short RNA probe. The critical importance of this short RNA sequence in mediating Rex function in vivo is supported by its complete conservation among all primate T-cell leukemia virus isolates.


Effects of altering palmitylation sites on biosynthesis and function of the influenza virus hemagglutinin. Mutagenesis studies indicated that the three cytoplasmic cysteines of the influenza virus A/Japan/305/57 hemagglutinin (HA) are all palmitylated, but to an unequal extent. Replacement of all three cysteines abolished palmitylation, but affected neither HA biosynthesis nor function. Palmitate was not required for HA to be incorporated into virions.


Renal colic in pregnancy.

Renal colic in pregnancy. Renal calculi are an infrequent but significant management problem during pregnancy. We reviewed all cases of renal colic occurring during pregnancy between 1979 and 1990 at Grace Hospital, a tertiary care obstetrical hospital in Vancouver, British Columbia. Of the patients 80 had a discharge diagnosis of renal colic and pregnancy during this 11-year period. Calculi were confirmed in 57 patients. Of the patients 66% were multiparous and 99% of the calculi occurred during either the second or third trimester. The most common symptom was flank pain seen in 89% of the patients, while greater than 95% displayed either microscopic or gross hematuria. Methods of radiographic diagnosis included ultrasonography and limited stage excretory urography. A total of 84% of patients passed stones spontaneously. Indications for urological or obstetrical intervention included persistent pain, sepsis, progressive hydronephrosis, solitary kidney or high grade obstruction. There were 37 procedures done in 23 patients. The most common procedure was placement of a ureteral stent. The complication rate associated with intrapartum intervention and stent passage in the 23 patients was 16%. All patients with a ureteral stent subsequently had spontaneous vaginal delivery without complication. A scheme for managing renal calculi in pregnancy is presented.


Intraoperative sonography of renal tumors. Intraoperative sonography (7.5 MHz. transducer with Doppler probe) was used to evaluate renal masses in 41 kidneys to determine its correlation with pathological findings. This intraoperative determination of the extent of the renal lesion prompted occasional changes in surgical management. We conclude that intraoperative sonography is most useful in partial nephrectomy, and helps to determine the extent of tumor, multicentricity, venous extension and associated cysts. These findings may not be appreciated with intraoperative, visual inspection of the kidney.


Clean intermittent catheterization and urinary diversion in the management of renal transplant recipients with lower urinary tract dysfunction. Renal transplant recipients with lower urinary tract dysfunction may be managed by urinary diversion or clean intermittent catheterization. To evaluate the comparative problems associated with each mode of therapy we studied 13 patients managed by clean intermittent catheterization (group 1, 6 patients) or urinary diversion (group 2, 7 patients). All 6 and 2 of 7 recipients in groups 1 and 2, respectively, had a neuropathic bladder. Mean followup was 3.7 +/- 1.5 (group 1) and 5.7 +/- 4.9 (group 2) years. Three allografts were lost in group 2 due to rejection (2) and death (1). Two patients each in groups 1 and 2 had febrile urinary tract infections requiring hospitalization and intravenous antibiotics. Complications related to urinary diversion developed in 4 patients in group 2. The serum creatinine at 1 and 3 years, number of hospital days per patient, total number of rejection episodes and number of patients employed in a full-time job following transplantation were similar in both groups. In summary, clean intermittent catheterization appears to have more morbidity in immunosuppressed transplant versus nontransplant patients but it may be preferable in renal transplant recipients due to the overall simplicity, positive psychological effect and comparable morbidity to other forms of management.


Complications of radical cystectomy and urinary diversion: a retrospective review of 675 cases in 2 decades. A retrospective review was performed on all 675 patients who underwent radical cystectomy and urinary diversion during 2 decades. Of the patients 197 were treated from 1969 to 1979 (group 1) and 478 were treated from 1980 until 1990 (group 2). The mean age of patients in group 1 was 56.7 years versus 64.2 years in group 2 (p < 0.001). The overall operative mortality rate in both groups was 2.5%. A total of 215 patients (31.9%) experienced postoperative complications (within 30 days of surgery). The morbidity rate was nearly identical between the 2 groups (32.0% for group 1 versus 31.8% for group 2, p = 0.962). Of note, however, there was a decreased incidence of wound infections and wound dehiscence among the patients in group 2 compared to group 1. Long-term complications occurred in 198 of the 675 patients (29.3%). At followup group 1 had a 35.5% incidence of long-term complications versus 26.8% in group 2 (p = 0.022). Most notably there was significant improvement in the incidence of ureteroenteric anastomotic strictures when comparing groups 1 (11.2%) and 2 (5.2%) (p = 0.006).


Tamm-Horsfall protein as a marker in interstitial cystitis. It has been suggested that immunohistochemical staining for Tamm-Horsfall protein in bladder epithelium may be a marker for interstitial cystitis. Of bladder biopsies from 14 interstitial cystitis patients only 3 demonstrated positive staining for Tamm-Horsfall protein within the mucosa, whereas 2 of 11 control biopsies showed positive Tamm-Horsfall protein results. In addition, staining in 4 ureteral specimens from interstitial cystitis patients and 4 controls was negative in all cases. An effort to detect this protein in enterocystoplasty specimens also showed a negative pattern. Our study does not confirm the Tamm-Horsfall protein as permeating either the bladder epithelium in interstitial cystitis or bowel mucosa in enterocystoplasty. This finding does not necessarily mean that these surfaces are not permeable to substances of smaller molecular size, nor does it define whether this is of importance in the etiology of interstitial cystitis. However, it does suggest that this technique does not allow detection of a marker for the disease.


Idiopathic reduced bladder storage versus interstitial cystitis. Idiopathic reduced bladder storage is a term we used to describe a group of patients who have subjective and objective evidence (by cystometrogram) of diminished bladder capacity without a demonstrable cause. We performed a prospective study comparing this condition with interstitial cystitis. We studied the clinical, urodynamic and histological features, and response to therapy in these 2 groups of patients. No statistical difference was found between the incidence of irritative bladder symptoms and/or suprapubic pain. Only minor differences were noted in the maximum cystometric capacity and incidence of bladder instability. Histological and immunofluorescent features were analogous. Also, the reduced bladder storage and interstitial cystitis patients responded similarly to bladder dilation and pharmacological therapy. Augmentation ileocystoplasty used in patients refractory to medical treatment produced comparable results in the short term. Based upon similar findings, it is likely that these 2 conditions represent the same disease entity with the only difference being the cystoscopic findings.


Treated history of noninvasive grade 1 transitional cell carcinoma. The National Bladder Cancer Group. A total of 178 patients with grade 1 noninvasive (stage Ta) bladder tumors followed from 1 to 10 years (median 58 months) was prospectively evaluated by cystoscopy, transurethral resection, mucosal biopsies, cytology, size and number of tumors at diagnosis, recurrences, progression in grade and stage, number of negative or positive cystoscopies and death from all causes. Histopathological and cytological studies were confirmed by a Central Pathology Laboratory using the criteria for grade 1 as described previously. Of the patients 122 (68.5%) had a single tumor. Three-quarters of the patients had tumors of less than 2 cm., 95% had mild or no urothelial dysplasia and 1 had positive cytology results. There were 419 recurrent tumors in 109 patients (61%). Patients with multiple tumors were at a significantly greater risk for recurrences (p < 0.001). Size of tumor significantly affected the rate of recurrence in the first 2 years after initial diagnosis in single tumor patients only. Of the multiple tumor patients 90% experienced a recurrence compared to 46% of the single tumor patients. Of the 1,112 cystoscopies performed in 122 single tumor patients 18% were positive, compared to 33% of the 686 cystoscopies performed in 56 multiple tumor patients. A total of 29 patients had a change in grade, 5 having grade 3 and 24 having grade 2 tumors. Progression to stage T1 occurred in 5 patients and to stage T2 or greater in 3. Of the 36 patients who died, 1 died of obstruction due to bladder cancer. Experimental evidence supports the opinion that the cells of stage Ta, grade 1 tumors are different in several ways from normal urothelium. There are little data to support the use of the term papilloma to describe stage Ta, grade 1 tumors without reservation. The data demonstrate that the tumor diathesis being expressed ceases with time and for unknown reasons. Multiple tumor patients with stage Ta, grade 1 disease might be included in chemotherapy trials only with stratification and a control arm of transurethral resection/fulguration alone.


Prognostic significance of mucosal aneuploidy in stage Ta/T1 grade 3 carcinoma of the bladder. In a prospective series of 71 patients with newly detected grade 3, stages Ta and T1 bladder carcinoma tumor characteristics, including the results of deoxyribonucleic acid (DNA) analysis as well as morphological and DNA characteristics of the grossly normal urothelium, were investigated and related to progression-free survival. The mean duration of followup was 57 months, with a minimum of 24 months. Of the 71 patients 24 underwent primary cystectomy, and 47 were conservatively treated with transurethral resection alone, or followed by instillation therapy or irradiation therapy. Of the cystectomy and conservatively treated patients 2 (8%) and 16 (34%), respectively, died of bladder carcinoma. Among the 47 conservatively treated patients tumor progression could not be predicted by the initial characteristics of tumor stage, papillary or nonpapillary growth, tumor multiplicity, tumor size, existence of 1 or multiple aneuploid cell populations, S phase value, carcinoma in situ and atypia or aneuploidy in the mucosal biopsies. Neither was progression predicted by the recurrence rate during year 1 of observation. However, a change to or persistent mucosal aneuploidy and a change to or persistent morphological abnormality of the mucosa during year 1 of observation were predictive for tumor progression (p = 0.001 and 0.045, respectively). When compared in stepwise regression analysis (Cox's proportional hazard model), DNA aneuploidy in the mucosa at 12 months after diagnosis was a highly significant predictor, whereas morphology added no further prognostic information. Therefore, progression is related to gross chromosomal abnormalities of the mucosa. High risk patients can be identified by evaluation of the grossly normal mucosa, which should be done as part of the initial diagnosis and during followup in conservatively treated patients with stages Ta and T1, grade 3 bladder carcinoma.


The value of immediate or early catheterization of the traumatized posterior urethra. A total of 16 patients with posterior urethral ruptures was treated with the aim of reestablishing urethral continuity immediately or early after injury. Followup ranged from 13 to 83 months (average 27). In all patients an emergency retrograde urethrogram demonstrated extravasation from the posterior urethra. Of the patients 13 were treated with a urethral catheter either immediately or within 1 to 5 weeks after injury. Three patients were treated with a suprapubic catheter alone after unsuccessful attempts at reestablishing urethral continuity and all 3 subsequently required urethroplasty for an obliterative stricture. These 3 patients were also impotent after injury. Of the 13 patients treated with a urethral catheter 8 had the catheter inserted either retrograde (2) in the emergency room or antegrade (6) in the operating room just after the injury, and in 5 the catheter was inserted transurethrally at cystoscopy within a mean of 3 weeks after injury. A total of 7 patients (54%) treated with urethral catheterization had a stricture during followup: 4 responded well to internal urethrotomy and 3 required simple dilation. Of 12 patients 5 (42%) became impotent after injury, while 1 was impotent before injury. No patient became incontinent. We conclude that careful urethral catheter realignment either immediately or within 5 weeks after injury is safe and obviates total urethral closure. Impotence may result from the severity of the injury and not from management with catheterization.


Penile extensibility: to what is it related? Penile extensibility is the difference between the length of the flaccid penis and the penis submitted to a maximal constant traction. This measurement has been proposed for use as a new potential diagnostic method to assess intracavernous fibrosis. To determine the value of this method we measured penile extensibility before and after removing the skin and cavernous tissue in 17 cadavers and 4 patients undergoing penile implantation. In all cases a cavernous tissue biopsy was performed and the percentages of the different structures were objectively quantified by computer analysis. The extensibility decreased with patient age but it was not influenced by removal of the skin and/or cavernous tissue. No correlation was observed between decreased extensibility and the increase in fibrotic elements in the penile tissue. Penile extensibility seems to reflect the elasticity of the tunica albuginea, which is the limiting factor and cannot be an expression of fibrosis of the corpora cavernosa.


Vascular impotence: focal or diffuse penile disease. Using computerized image analysis the percentage of smooth muscle fibers was measured in several biopsies of the penis from 10 cadavers and 5 patients with vascular impotence. No significant difference was observed between the proximal and distal areas, and/or between the peripheral and central areas in 1 corpus cavernosum, and between the 2 corpora cavernosa for each patient or cadaver. The percentage of smooth muscle fibers was less in impotent patients in comparison with the cadavers, in which the erectile status was not known, and this reduction occurred throughout both cavernous bodies. Vascular impotence is a diffuse penile disease. Therefore, a cavernous body biopsy can be used to study the penile structure in the assessment of vascular impotence.


Preliminary results with the nitric oxide donor linsidomine chlorhydrate in the treatment of human erectile dysfunction. Recent experimental studies showed an important role of endothelium derived relaxing factor for cavernous smooth muscle relaxation. Since nitric oxide seems to account for the biological actions of endothelium derived relaxing factor, a study was done to examine a possible role of the nitric oxide donor linsidomine chlorhydrate (SIN-1) in the treatment of erectile dysfunction. To determine a therapeutically useful dose 0.1, 0.2, 0.5 and 1 mg. SIN-1 were injected intracavernously in patients with erectile dysfunction. Each dose was given to 2 patients. Then, 63 patients received 1 mg. SIN-1, including 7 who had prolonged erections to minimal doses of papaverine plus phentolamine and 4 who did not respond with a full erection to other pharmacological agents. Intracavernous injection of SIN-1 induced a dose-dependent erectile response by increasing the arterial inflow and relaxing cavernous smooth muscles. Of the patients 29 had a full, 21 an almost full and 13 a moderate erection to 1 mg. SIN-1. There were no systemic or local side effects. In the patients with prolonged erections to papaverine plus phentolamine the mean duration of a full erectile response to SIN-1 was 57 minutes. Compared to the responses to a papaverine (15 mg./ml.) and phentolamine (0.5 mg./ml.) mixture, the erection induced by SIN-1 was superior in 10, comparable in 47 and inferior in 6 patients. Our data suggest a possible role for SIN-1 in the treatment of erectile dysfunction. Possible advantages may be that erection is induced by a mechanism similar to that occurring physiologically, a decreased risk of inducing prolonged erections and low therapy costs.


Volume-dependent intracavernous hemodilution during pharmacologically induced penile erections. The change in the cavernous hematocrit following induction of pharmacological erection by an intracavernous injection of papaverine hydrochloride was documented in normal controls and patients with impotence. Blood samples taken from the penile cavernosa showed a significantly lower hematocrit compared to the systemic venous blood in all normal subjects. The decrease in the cavernous hematocrit was attributable to dilution of the cavernous blood pool by the injected volume of the drug, since this was not observed in erections produced by visual sexual stimulation. It appears that a restriction of the cavernous venous outflow in response to papaverine injection causes sequestration of the diluted blood in the cavernous compartment. The degree of cavernous hemodilution was found to aid in the differential diagnosis and was especially valuable in differentiating patients with arteriogenic impotence from those with venous leakage.


Early experience with the controlled girth and length expanding cylinder of the American Medical Systems Ultrex penile prosthesis. A prospective study was done to evaluate length expansion characteristics of the AMS Ultrex penile prosthesis cylinder intraoperatively and in the early postoperative period. A total of 50 patients 30 to 74 years old (mean age 55.2 years) underwent implantation of this device. Intraoperative changes in the pubis to mid glans distance between deflation and inflation ranged from 1 to 4 cm. (mean 1.9). Postoperative followup measurements were obtained in 46 patients at 1 to 15 months (mean 4). There was a 1 cm. decrease in the amount of postoperative length expansion for 6 patients, while length expansion increased by 1 cm. for 12 and for 28 the postoperative length expansion was the same as the intraoperative length expansion.


Varicocele treatment: prospective randomized trial of 3 methods. This study was done to evaluate the surgical results and the impact on fertility potential of 3 methods of varicocele treatment. Consecutive varicocele patients with primary or secondary infertility were randomly assigned to 3 treatment groups. Of the patients 36 underwent percutaneous embolization, 55 high ligation of the internal spermatic vein and 28 transinguinal simultaneous ligation of the internal and external spermatic veins. The transinguinal ligation proved to be safe. There was no difference in pregnancy rates but the seminal variables showed a slight improvement with statistical significance only in the 2 open surgical methods. There were no surgical failures in the transinguinal group as opposed to the other 2 techniques. Transinguinal ligation of the internal and external spermatic veins may be recommended as the primary treatment for varicocele. This technique also seems to be the procedure of choice when repeat intervention is required for failure of high ligation or embolization.


A test for the identification of relevant sympathetic nerve fibers during nerve sparing retroperitoneal lymphadenectomy. We present a new intraoperative test for the identification of sympathetic nerve fibers relevant to ejaculation during nerve sparing retroperitoneal lymphadenectomy in patients with nonseminomatous testicular tumors. Electrostimulation of specific isolated postganglionic nerve fibers resulted in intraoperative ejaculation in 9 of 11 patients. Ejaculation was without tumescence in a noneruptive manner and was reproducible in most cases upon repeated stimulation. In 6 patients ejaculation resulted after stimulation of the L3 fiber and in 1 each after stimulation of the L1, L2 and hypogastric plexus. The test seems to be particularly useful in post-chemotherapeutic dissections or otherwise extended dissections when sparing of irrelevant fibers is to be avoided.


Transurethral surgery using intravesical bupivacaine and intravenous sedation. We report our experience using intravesical 0.5% bupivacaine as a topical anesthetic along with intravenous fentanyl and midazolam sedation to perform a variety of transurethral procedures in 78 patients. We achieved adequate pain control in all patients and observed no anesthetic complications. Use of this combination of intravesical topical anesthesia and intravenous sedation provided safe, adequate anesthesia to our patients undergoing various transurethral procedures in an outpatient clinic setting.


Ambulatory monitoring of bladder pressure in low compliance neurogenic bladder dysfunction. Upper tract dilatation is an important complication of neurogenic bladder dysfunction. Risk factors include incomplete bladder emptying with large residual volumes of urine and high tonic increases in bladder pressures during artificial filling. However, on natural bladder filling many of these patients do not have high tonic increases in detrusor pressures. We compared conventional urodynamic studies with ambulatory monitoring during natural bladder filling in 66 patients with low compliance neurogenic bladder dysfunction. There were marked differences in the tonic increase in bladder pressure during filling and in compliance during artificial bladder filling compared with ambulatory monitoring. Faster filling rates during artificial filling resulted in greater end filling pressures and lower compliance but the lowest increases in bladder pressure were found during ambulatory monitoring with natural bladder filling. During natural bladder filling significantly more patients had phasic changes in detrusor pressure; a high intensity of phasic activity during ambulatory monitoring correlated with high end filling pressures during artificial bladder filling. Upper tract dilatation was associated with large volumes of residual urine, high resting bladder pressures and low bladder compliance on filling at 100 ml. per minute. However, upper tract dilatation was most strongly associated with high intensity phasic pressure activity during natural bladder filling in combination with high residual urine volumes and high resting bladder pressures. On multivariate statistical analysis the intensity of phasic pressure activity during ambulatory monitoring was the best discriminator between patients with dilated and normal upper tracts. Our study has highlighted important differences in the results obtained by artificial filling cystometry and ambulatory monitoring during natural bladder filling. In particular, high increases in pressure did not occur during natural bladder filling, apparently being replaced by phasic activity. Within this group of patients who had the high risk factor of low bladder compliance measured during artificial bladder filling, a combination of greater residual urine volumes, greater resting pressures and greater phasic activity during natural bladder filling was found in patients with upper tract dilatation.


A quantitative histological analysis of the dilated ureter of childhood. A quantitative histological study of the dilated ureter of childhood was performed on 26 ureters. The specimens were from 15 male and 11 female patients 10 days to 12 years old (mean age 2.0 years). A color image analysis system was used to examine and compare collagen and smooth muscle components of the muscularis layers to normal control ureters of similar age. In comparing primary obstructed (12) to primary refluxing (14) megaureters and control ureters (6), there was a statistically different collagen-to-smooth muscle ratio (p < 0.001) between the primary obstructed and primary refluxing megaureter groups. For patients with primary refluxing megaureter there was a 2-fold increase in the tissue matrix ratio of collagen-to-smooth muscle when compared to patients with primary obstructed megaureter. In the primary obstructed megaureters the amount of collagen and smooth muscle was not statistically different from controls (p > 0.01). The increased tissue matrix ratio of 2.0 +/- 0.35 (collagen-to-smooth muscle) in the refluxing megaureter group compared to 0.78 +/- 0.22 in the obstructed megaureter group and 0.52 +/- 0.12 in controls was found to be due not only to a marked increase in collagen but also a significant decrease in the smooth muscle component of the tissue. Primary obstructed and normal control ureters had similar quantitative amounts of smooth muscle with 60 +/- 5% and 61 +/- 6%, respectively, while refluxing megaureters had only 40 +/- 5% smooth muscle. The percentage collagen was 36 +/- 5 in the obstructed megaureter group and 30 +/- 5 in controls, with refluxing megaureters having 58 +/- 5% collagen on analysis. Our findings emphasize the significant differences in the structural components (collagen and smooth muscle) of the dilated ureter of childhood, and provide us with further insight into the pathological nature of these dilated ureters and their surgical repair.


Prenatal diagnosis of the megacystis-megaureter association. A total of 11 male neonates with hydronephrosis and a large bladder in utero had the megacystis-megaureter association. Prenatal ultrasound findings included bilateral hydroureteronephrosis; a large, smooth, thin-walled bladder, and normal volume of amniotic fluid. Postnatal studies demonstrated grades 4 to 5 bilateral reflux, a large bladder without obstruction and in 2 infants nonfunction of a kidney or renal moiety. Of 7 infants initially managed nonoperatively 5 have undergone surgery due to persistent reflux or breakthrough urinary infections. The prenatal diagnosis of the megacystis-megaureter association can be suspected with reasonable accuracy. Prompt postnatal continuous antibiotic prophylaxis and uroradiological confirmation allow for nonemergency management of this condition with excellent results.


Endopyelotomy associated ureteral necrosis: complete ureteral replacement using the Boari flap. A case of an adolescent who sustained necrosis of the entire ureter after attempted endopyelotomy for congenital ureteropelvic junction obstruction is presented. Successful reconstruction of a neoureter was performed easily with the Boari bladder flap coupled with nephropexy and a psoas hitch. Although repair of upper ureteral injuries with the Boari flap has been described in the literature, to our knowledge its use in the pediatric population has not. Our case exemplifies how the Boari flap repair is particularly suitable in children for bridging significant segments of injured ureters, not just the lower third.


Influence of preoperative bladder capacity and compliance on the outcome of artificial sphincter implantation in patients with neurogenic sphincter incompetence. We performed a retrospective study of 23 patients with neurogenic sphincteric incompetence who had undergone implantation of an artificial urinary sphincter to determine if bladder capacity and compliance as determined by cystometrography could predict the need for enterocystoplasty. Study criteria were neurogenic sphincteric incompetence, no previous operations on the lower urinary tract, and performance of preoperative and postoperative cystometrography. Patients were 5 to 17 years old at implantation. Incontinence was caused by myelomeningocele (18 patients), sacral agenesis (3) and spinal cord tumor (2). The 8 patients for whom preoperative cystometric bladder capacity was greater than 60% of the expected capacity for age have been followed for a mean of 60 months. All 8 patients are continent and none required enterocystoplasty. Preoperative bladder compliance exceeded 2 ml./cm. water in all patients (group 1). Of the 15 patients for whom preoperative cystometric bladder capacity was less than 60% of the expected value (group 2, small bladders) 8 followed an average of 72 months had a compliance greater than 2 ml./cm. water and have done well without bladder augmentation. In contrast, 7 patients in this group (46%) required enterocystoplasty: 6 for persistent or recurrent incontinence and 1 for upper tract changes. The average interval between artificial sphincter placement and enterocystoplasty was 14 months. Patients with a small bladder that required augmentation had a preoperative bladder compliance of less than 2 ml./cm. water. We conclude that small bladder capacity, as determined by cystometrography in patients with neurogenic sphincteric incompetence but a bladder compliance of less than 2 ml./cm. water predicts the future need for bladder augmentation. In all other patients, with good medical treatment and followup, the possible adverse effects of a small capacity bladder can be prevented or corrected. With this strategy we have been able to avoid enterocystoplasty with its attending potential complications in 70% of our patients with neurogenic incontinence and favorable urodynamics regardless of preoperative cystometric bladder capacity.


Adventitious knots in urethral catheters: report of 5 cases. Urethral catheterization with 5 or 8F feeding tubes in 5 boys was complicated by stochastic knotting within the bladder thereby impeding removal. The common factor in these patients was insertion of excessive length within the bladder. Percutaneous endoscopic retrieval was done successfully in 1 child. This technique may avoid urethral trauma associated with catheter removal, particularly in younger boys.


Radiographic and serologic correlates of azotemia in patients with posterior urethral valves. A retrospective statistical analysis with a minimum followup of 10 years was done on 102 patients who presented in early childhood with posterior urethral valves. All patients were treated with initial bladder drainage. Factors correlating with the development of renal failure were evaluated. No patient with a normal kidney on 1 side had renal failure. Calculated glomerular filtration rate was significantly higher in patients who retained adequate renal function (80.7 +/- 17.8 ml. per minute per 1.73 m.2), as compared with those who had renal failure (18.6 +/- 9.6 ml. per minute per 1.73 m.2). Patients with renal failure also had a significantly higher serum creatinine level at stabilization (2.0 +/- 0.8 mg./dl.) than those who had adequate renal function (0.5 +/- 0.2 mg./dl.). The presence of bilateral high grade vesicoureteral reflux, hydronephrosis and nonfunction was significantly higher in patients with renal failure. Patient age at presentation was not a significant factor. These data represent a guideline for the prognosis and management protocols of infants with dilated upper urinary tracts and posterior urethral valves.


Urethral stricture in children: treatment by urethroplasty with bladder mucosa graft. Urethral strictures in children, which are not frequent, often require urethroplasty when dilations and/ or urethrotomies have failed. A bladder mucosa graft was used successfully for urethral reconstruction to treat posterior hypospadias. We describe our experience with a bladder mucosa graft during urethroplasty for acquired urethral strictures in 8 children. Urethral strictures secondary to the treatment of hypospadias were excluded. Bladder mucosa was used successfully as an onlay or patch graft urethroplasty in 7 patients. One patient had a tubularized graft with secondary stenosis treated successfully by dilation.


Onlay island flap urethroplasty: variation on a theme. The onlay island flap urethroplasty is useful in patients with distal, mid shaft and proximal hypospadias who have a well developed urethral plate and exhibit little or no curvature after release of chordee. The technique described has 2 main features: 1) use of the entire inner prepuce of the foreskin facilitates its mobilization and protects the vascular pedicle, and 2) additional soft tissue coverage is gained by removing the excess preputial mucosa not used for the neourethra. This extra soft tissue covering of the suture lines should prevent fistula formation. During the last 4 1/2 years the onlay island flap has been used for repair of hypospadias in 61 patients. The complication rate (6%) is low and compares favorably with other techniques. Further refinements in the onlay flap hypospadias repair should decrease the complication rate and widen its applicability.


Penoscrotal transposition with hypospadias: 1-stage repair. A 1-stage surgical repair of penoscrotal transposition with hypospadias is described. The basic principles are correction of hypospadias with the best vascularized island penile skin flap used for a new urethra and 2 vascularized sliding skin flaps used for reconstruction of the penile skin, and transposition of the penis to the suprascrotal position in the area of the mons pubis, with mobilization plus midline testicular fixation (inter-orchiopexy) and scrotoplasty. This technique was applied in 42 patients 2 to 9 years old between 1986 and 1991. The complications were 2 urethral stenoses on the proximal anastomosis (1 was treated successfully by urethrotomy and 1 by an open operation) and 2 fistulas (successfully treated by surgery), while 2 patients required additional correction of penoscrotal transposition.


Intraoperative spermatic venography during varicocele surgery in adolescents. Intraoperative internal spermatic venography performed immediately following varicocele ligation in the adolescent has been touted as reducing varicocele persistence rates. Previously published data corroborate this statement with low persistence rates. Other series in which venography was not performed report a failure rate of 9 to 30%. During a 5-year period a total of 64 varicocele ligations was performed in 62 male adolescents at our institution. Followup postoperatively revealed an overall varicocele persistence rate of 9%. All patients had intraoperative internal spermatic venography on the affected side. Of 64 venograms 16% had shown collateral drainage that, if not ligated, may have resulted in varicocele persistence. These cases accounted for only 1 of the persistent varicoceles. Additionally, venograms had demonstrated filling of the ipsilateral external iliac vein in 8% of the cases. Despite the fact that no attempt was made to ligate these collaterals, none of these patients had a persistent varicocele. After varicocele ligation 30 of 62 patients were followed long enough to evaluate for testicular catch up growth. Of these 30 patients 24 demonstrated an average relative increase in left testicular volume of 17%. These data support routine intraoperative internal spermatic venography while performing varicocele ligation in the adolescent.


Endocytosis of calcium oxalate crystals and proliferation of renal tubular epithelial cells in a patient with type 1 primary hyperoxaluria. A patient with primary hyperoxaluria who received a liver-kidney transplant is presented. A postoperative renal biopsy showed apparent tubular cell endocytosis of calcium oxalate crystals and cell proliferation, indicating that renal epithelial cells do not perceive urinary crystals as inert. Such cellular responses to crystals may have a role in nephrolithiasis.


Arteriovenous fistula complicating endopyelotomy. We report a case of arteriovenous fistula and pseudoaneurysm formation following endopyelotomy. Presentation, successful management with interventional radiology techniques, and the relationship between variant renal artery anatomy and endopyelotomy are discussed.


The management of injuries to the urethra, bladder or vagina encountered during difficult placement of the artificial urinary sphincter in the female patient. Between 1977 and 1989, an artificial urinary sphincter was implanted in 57 female patients. In 6 patients inadvertent intraoperative injuries to pelvic organs occurred, 5 of whom had a history of an average 2.8 previous operations for incontinence (range 2 to 4). The remaining patient was a 16-year-old girl with primary internal sphincter incompetence. All 6 patients presented with total incontinence. Intraoperative injury included 4 women who sustained vaginal perforations, while 1 had an anterior bladder perforation and in 1 the urethra was entered. Mechanisms of injury were sharp perforation of structures adherent to the pubis and blunt tears of distorted urethrovaginal tissues. Primary closures of the urethra, bladder and vaginal defects followed by insertion of the artificial urinary sphincter were accomplished successfully. Postoperative management included vaginal antiseptic packs, appropriate antibiotics and delayed sphincter activation. Of the 6 patients 5 remained dry after initial placement with a mean followup of 32 months (range 7 to 77). The remaining patient required replacement with a higher pressure balloon and a smaller cuff, which resulted in complete continence.


Use of a polytetrafluoroethylene tube graft as a circumferential neotunica during placement of a penile prosthesis. Various techniques have evolved for augmentation of the tunica albuginea in cases when tunical tissue has been found deficient during penile prosthesis implantation. An intraoperative situation occurred in which the size of the tunical defect did not permit primary tunical closure or allow for approximation by placement of a tunical patch graft. In this case, to ensure satisfactory insertion of a penile prosthesis a polytetrafluoroethylene (Gore-Tex) tube graft was used as a circumferential neotunica.


Epididymo-orchitis developing as a late manifestation of intravesical bacillus Calmette-Guerin therapy and masquerading as a primary testicular malignancy: a report of 2 cases. In 2 cases epididymo-orchitis, indistinguishable from a testicular tumor, developed as a late (15 and 34 months, respectively) complication following use of Tice strain bacillus Calmette-Guerin for treatment of superficial bladder carcinoma. In each instance the lesion was asymptomatic and ultrasonography demonstrated a complex scrotal mass. Inguinal orchiectomy was performed for diagnosis and therapy. The importance of obtaining a mycobacterial culture for further therapy planning is stressed.


Prostatic abscess due to Candida tropicalis in a nonacquired immunodeficiency syndrome patient. We report a case of prostatic abscess due to Candida tropicalis in a nonacquired immunodeficiency syndrome patient with diabetes. The diagnosis and management are discussed, and the literature is reviewed.


Papillary adenocarcinoma in a seminal vesicle cyst associated with ipsilateral renal agenesis: a case report. Cysts and tumors of the seminal vesicle are uncommon, and their coexistence is extremely rare. We report a case of multiple papillary tumors inside a seminal vesicle cyst associated with ipsilateral renal agenesis in a 17-year-old man. Surgical excision of the cyst and tumors was performed without any morbidity and histology revealed well differentiated papillary adenocarcinoma.


Effect of PSK and its subfractions on peripheral blood lymphocytes mediated cytotoxicity against urinary bladder tumor cells. Our previous studies have indicated that the protein-bound polysaccharide Kreha (PSK) enhances the cytotoxic activity of peripheral blood lymphocytes (PBL) against the T24 human urinary bladder tumor cell line in patients with bladder tumor. Since PSK consists of a mixture of various kinds of protein-bound polysaccharides, the present study was designed to examine which subfractions of PSK mediated the enhancement of cytotoxicity. When PSK was separated according to size, treatment of PBL with the 50 kilodalton (kd) or less fraction killed T24 cells more efficiently than unfractionated PSK-treated PBL. The higher molecular weight fractions did not enhance killing above the control level. PSK was fractionated on a diethylaminoethyl (DEAE)-cellulose column to obtain a protein rich fraction that absorbed onto the column and a polysaccharide rich fraction that did not. PBL treated with the polysaccharide rich fraction were able to kill T24 cells more effectively than unfractionated PSK-treated PBL. The protein rich fraction had no effect on the killing. Further fractionation of the polysaccharide rich fraction was performed by differential precipitation with ammonium sulfate. PBL treated with the precipitated fraction at 70-80% saturation (PSK Fraction D) enhanced cytotoxicity equal to that of the polysaccharide rich fraction. Treatment of PBL with the other fractions did not augment the cytotoxicity. These enhancement by PSK fractions were observed in healthy donors and also in patients with bladder tumor. An increase of the proliferative response of PBL to PSK Fraction D as well as unfractionated PSK was observed. Treatment of PBL with PSK Fraction D had no effect on the proportion of PBL binding to T24 cells, thus suggesting a post-binding effect. The structure of PSK Fraction D as inferred from the results of methylation analysis was mainly an alpha-glucan. These results demonstrate that PSK mediated enhancement of cytotoxicity and proliferation of PBL may be largely due to an alpha-glucan of less than 50 kd.


Impaired immune response by isoniazid treatment during intravesical BCG administration in the guinea pig. At present, isoniazid (INH) is being used prophylactically to reduce the side effects of intravesical BCG therapy for superficial bladder cancer, although it is not clear whether or not this reduces the antitumor efficacy of BCG. In this study the impact of INH treatment on the immune response after repeated intravesical BCG administration was investigated in guinea pigs. INH was given on the 3 days around each BCG instillation. We found that the administration of INH severely impaired the immunological effects of BCG. The induction of mononuclear cell infiltration in the bladder wall was reduced. Enlargement of the regional lymph nodes (weight and number of cells), and increase of MHC Class II expression on the lymph node cells, normally observed after intravesical BCG administration, were inhibited by INH. Systemic immunity, measured by the DTH reaction in the skin to PPD, was also diminished due to the combined treatment of BCG with INH. When INH was administered during the last 4 of 6 BCG instillations, the immune response to BCG was still impaired. A five-fold increase of the dose of BCG did not overcome the effect of INH. INH probably did not exert a direct suppression of the immune system of the guinea pig as the DNCB skin reactivity was not influenced. Although INH concentrations in the urine were high at the onset of the instillation, in vitro experiments indicated that the effect of INH may not be caused by killing of the BCG organisms shortly after application in the bladder. In conclusion, our data in guinea pigs suggest that the use of INH may impair the immune response to intravesical BCG. As this response may be important for the antitumor effect of BCG, urologists should be cautious with the prophylactic use of INH. The influence on the antitumor efficacy is now investigated in man.


Expression of adhesion molecules by bladder cancer cells: modulation by interferon-gamma and tumour necrosis factor-alpha. The constitutive expression by eight human bladder cancer cell lines of the cell adhesion molecules intercellular adhesion molecule-1 and intercellular adhesion molecule-2 was studied using monoclonal antibody probes in conjunction with flow-cytometry. Tumour lines of low grade (G1) did not constitutively express intercellular adhesion molecule-1, rather they were found to express intercellular adhesion molecule-2. The G2 cells expressed no intercellular adhesion molecule-2, however, a low percentage did express intercellular adhesion molecule-1. High grade cells (G3) only expressed intercellular adhesion molecule-1 on their cell surface but at higher levels than the G2 cell line. Exposure of the bladder cancer cell lines to interferon-gamma induced and augmented the expression of intercellular adhesion molecule-1 by all except one of the cell lines (UMUC3). Intercellular adhesion molecule-2 expression remained unaltered. The modulation of intercellular adhesion molecule-1 expression was dependent on the concentration of interferon-gamma and the duration of stimulus. De novo intercellular adhesion molecule-1 expression, induced by interferon-gamma, was rapid (< 4 hours) with only a short period of stimulation being required (< 10 seconds). The rapid increase in expression of intercellular adhesion molecule-1 required de novo protein synthesis and was not the result of release of intercellular adhesion molecule-1 from an intracellular pool. Interferon-gamma and tumour necrosis factor-alpha were found to act synergistically in the induction and augmentation of intercellular adhesion molecule-1 expression. Optimal induction occurred with 10 Uml-1 of both molecules. These results suggest a correlation between constitutive adhesion molecule expression and the histopathological grade of the tumour. The implications of these findings for Bacillus Calmette Guerin and interferon-gamma immunotherapy of bladder cancer is discussed.


Diabetes mellitus impairs neurogenic and endothelium-dependent relaxation of rabbit corpus cavernosum smooth muscle. The effect of alloxan-induced diabetes on the reactivity of corporeal nerves, endothelium and smooth muscle was studied in the New Zealand white rabbit. Fifteen rabbits were randomly divided into treated (n = 6) and control (n = 9) groups. The treated group was maintained for 6 weeks. Two control groups were studied. One control group (n = 3) was maintained for 6 weeks as littermate controls for diabetic group. The second control group (n = 6) was not maintained but was weight matched with the 6 week diabetic group. The reactivity of corpus cavernosum tissue from the diabetic animals and the control animals was studied in organ chambers. When tissue contraction was produced with phenylephrine for the study of relaxation to various stimuli, the tension induced was similar in the diabetic and the control groups. Relaxation of corpus cavernosum tissue to electrical stimulation of autonomic nerves as well as relaxation to the endothelium-dependent vasodilator acetylcholine were comparably unaffected in the weight matched and littermate control groups while significantly inhibited in the diabetic group. Treatment of the corporeal tissue with the cyclooxgenase inhibitor indomethacin enhanced the relaxation to electrical stimulation and to acetylcholine in the control and in the diabetic groups but did not improve the significant difference in relaxation between the two groups. Relaxation of corporeal tissue to endothelium-independent vasodilators, papaverine and nitroprusside was similar in the control groups and the diabetic groups. It is concluded that diabetes impairs neurogenic and endothelium-mediated relaxation of rabbit corpus cavernosum smooth muscle. These findings are comparable to those described in corpus cavernosum tissue from diabetic men, showing the validity of this experimental animal model. The mechanism for the nerve or endothelial dysfunction does not appear to involve alteration in cyclooxygenase products of arachidonate or the ability of the corporeal smooth muscle to relax via a cGMP-dependent mechanism. Since nitric oxide has been shown to act as the nonadrenergic noncholinergic neurotransmitter as well as endothelium-derived relaxing factor (EDRF) of the trabecular smooth muscle, it is possible that impairment of neurogenic and endothelium-dependent relaxation due to diabetes is mediated by alteration in the synthesis or availability of nitric oxide in corporeal tissue.


Microbial adhesion and biofilm formation on ureteral stents in vitro and in vivo. Thirty ureteral stents, inserted for 5 to 128 days following extracorporeal shock wave lithotripsy, were examined for the presence of bacterial biofilms. Of these, 90% had adherent pathogens (44% mixed organisms) on the stents, 45% of which were present in low numbers (10(1)-10(2) per 1 cm3 section) and 55% were in small and large microcolony biofilms (> 2 x 10(2)-10(7)). The organisms were recovered from the stents even though urine culture was only positive in 27% of patients. Of the organisms isolated, 77% were Gram positive cocci, 15% Gram negative rods and 8% Candida. No blockage of the stents occurred. All of the patients had received antimicrobial therapy post-insertion, and in 15 cases biofilms were found while on treatment. None of the patients received therapy for urinary tract infections while the stent remained in place. In vitro experiments demonstrated the ability of Escherichia coli, Proteus mirabilis, Staphylococcus epidermidis and Enterococcus faecalis uropathogens to adhere and form biofilms on ureteral stents within 24 hours. Clearly, bacterial biofilms do occur on ureteral stents and urinary culture may not detect their presence. The high recovery rate of Gram positive organisms may indicate a preferential adhesion to the biomaterial surface. The findings also indicate that unlike biofilm formation on many other prosthetic implants, colonization with Gram positive organisms on ureteral stents does not necessarily coincide with the development symptomatic infection.


Allelic loss of chromosome 17p in urothelial cancer: strong association with invasive phenotype. Allelic loss of chromosome 17p with a mutated p53 gene on the remaining allele has been observed in various kinds of human cancers. To examine the significance of allelic loss of chromosome 17p in human urothelial cancer with special attention to the clinicopathological features, 49 tumors with various stages and grades from 43 cases (35 bladder cancers and 8 renal pelvic or ureteral cancers) were examined for loss of heterozygosity using 5 polymorphic probes on chromosome 17p. Thirty-seven cases were informative, and allelic loss of chromosome 17p was observed in 15 (41%) of them. In bladder cancers, the loss of 17p was observed with significantly higher frequency (p < 0.01) in cases with invasive (> or = pT2) tumors (7/10, 70%) than in cases with superficial (pTa or pT1) tumors (4/21, 19%). In renal pelvic or ureteral cancers, none of 2 superficial tumors and all of 4 invasive tumors showed the allelic loss. As to tumor grade, the allelic loss was observed in 1/9 (11%) for grade 1 cases, 6/18 (33%) for grade 2 cases, and 8/10 (80%) grade 3 cases (grade 1 versus 3, p < 0.01; grade 2 versus 3, p < 0.05). On the other hand, examination of clinical features, such as primary tumor site, tumor multiplicity or previous history of urothelial cancer did not significantly influence the frequency of the allelic loss. Our results suggest that the allelic loss of chromosome 17p is strongly associated with invasive phenotype in urothelial cancer. The results further indicate that the 17p deletion may represent a new genetic marker of malignant potentials in urothelial cancers.


Urinary bladder function in the tight-skin mouse. Tight-skin mice develop hypertrophy of connective tissue and tendons, associated with increases in collagen concentration in skin, heart, lungs, and tail. The bladders from these mice have not previously been examined. Because of the purported importance of collagen in bladder wall structure and compliance, we examined collagen content, micturition characteristics, and length-tension relationships in bladders from tight-skin mice. Bladder collagen content and concentration were approximately 70% greater in 5-6 month tight-skin mice than age-matched controls, but bladder mass, protein content, and protein concentration were similar. Tight-skin mice urinated larger volumes more frequently during the light cycle, and the functional bladder capacity appeared to be greater than that of controls. There was a small shift to the right of the passive length-tension curves of bladder strips from tight-skin mice, but the shift was not statistically significant. The magnitude of active tension development was the same. The data suggest that bladder collagen concentration does not necessarily determine bladder capacity or compliance. It is suggested that other factors, such as the ratio of collagen subtypes or the collagen:elastin ratio may have more importance for the maintenance of bladder distension.


Comparison of urinary bladder function in 6 and 24 month male and female rats. Micturition characteristics, collagen composition, and in vitro urinary bladder strip contractility were examined in young adult (six month) and old (24 month) male and female Fischer 344 rats. Although young female rats consumed significantly less water than young males, there were no differences in volumes of urine excreted. Old females excreted significantly more urine than old males, but there were no differences in volumes of water consumed. Old male rats had similar micturition frequencies during the light and dark cycles, in contrast to females and young males, where the number of micturitions during the dark cycle was significantly greater than those during the light cycle. The mean and maximal micturition volumes were significantly greater in old males compared to young males and old females during both the light and dark cycles. Bladders from female rats weighed significantly less than bladders from males of the same age, and the bladders from young rats weighed less than those of old rats. The protein and collagen concentrations were significantly less in bladder bodies from young females than old females. The amount of collagen resistant to digestion by Pronase, and thus thought to be cross-linked, was significantly greater in bladders from old rats compared to young. No differences between groups were found in the contractile responses of bladder base strips. There were trends for the absolute contractile responses of bladder body strips from old males to field stimulation, carbachol, ATP, and KCl to be larger than the other groups, and for strips from the young females to be smaller. The responses of strips from young females to field stimulation and KCl were significantly less than those of young males or old females, and responses to 10(-3) M ATP were less than those of old females. Responses of strips from old males to 60 mM KCl were significantly greater than those of young males. The differences in contractility could be attributed to the differences in strip mass. It appears, therefore, that urinary bladder function in male and female rats is unaffected by increasing age between 6 and 24 months.


The detection and characterization of vesicoureteral reflux in the child. Voiding cystourethrography or radionuclide cystography should be performed in the child in whom it is important to know whether reflux is present. Voiding cystourethrography is more accurate in characterizing and grading reflux, and monitoring is intermittent. Conversely, radionuclide cystography uses a lower radiation dose and its continuous monitoring leads to fewer false negative results but its ability to characterize reflux is poor. The performance of voiding cystourethrography or radionuclide cystography to detect reflux is an art as well as a science. These tests are done best by those who are experienced and interested in imaging the urinary tract of the child. At our hospital we perform voiding cystourethrography in all young children with urinary tract infection to detect and precisely characterize reflux to enable intelligent planning of management. In older patients, when it is less likely that reflux is present but it is still desirable to ensure that reflux is not occurring, radionuclide cystography is performed. If reflux is present, then a decision is made on an individual basis as to whether additional characterization by voiding cystourethrography is needed. Radionuclide cystography is also used for family screening, for periodic followup of reflux being managed nonoperatively and to ensure that reflux has been eliminated after antireflux surgery.


Characteristics at entry of children with severe primary vesicoureteral reflux recruited for a multicenter, international therapeutic trial comparing medical and surgical management. The International Reflux Study in Children. A total of 532 children, 401 from Europe and 131 from the United States, was recruited into an international multicenter study comparing the results of medical and surgical treatment of children with international grade III or IV vesicoureteral reflux and urinary tract infection. Patient age was less than 11 years, glomerular filtration rate was greater than 70 ml. per minute per 1.73 m.2 and there was no obstruction, renal malformation, previous urinary tract surgery or neuropathic bladder. A total of 80 children had less than grade III vesicoureteral reflux on a second pre-entry cystourethrogram required by the European protocol, and they were followed separately as a sideline group. At entry the age distribution, history of urinary tract infection and proportion of children with grade IV vesicoureteral reflux were similar in Europe and the United States. Of the European and United States children 48% and 54% had renal scarring, and 17% and 14% had parenchymal thinning, respectively. In each group renal length and planimetric area were normal in two-thirds and 5% had small kidneys (<2 standard deviations from normal). Differences included sex distribution (24% boys in Europe and 11% in the United States group), the proportion of children in whom vesicoureteral reflux was previously known (18% Europe and 69% United States) and the proportion of children with bilateral reflux (77% Europe and 57% United States). Randomization and stratification for treatment were successful.


Infection pattern in children with vesicoureteral reflux randomly allocated to operation or long-term antibacterial prophylaxis. The International Reflux Study in Children. A total of 306 children with grade III to IV vesicoureteral reflux (international classification) and a history of documented urinary tract infection was randomized into medical (155 patients) or surgical (151 patients) treatment arms in the European portion of the International Reflux Study in Children. Children treated medically were maintained on prophylactic antibacterials as long as the reflux persisted, while those treated surgically were covered prophylactically until followup studies at 6 months postoperatively demonstrated the reflux to be corrected. Standard definitions for bacteriuria were used, and the distinction was made clinically among acute pyelonephritis, cystitis and asymptomatic bacteriuria, supported in many instances by additional laboratory testing. Urine was cultured after 3 months and whenever suspicious symptoms occurred. Urinary tract infections developed during the first 5-year followup period in 59 patients (38%) in the medical group and in 59 (39%) in the surgical group but the incidence of pyelonephritis was higher in the medical group (21%) than in the surgical group (10%) (p < 0.01). Pyelonephritis often followed catheterization or cystoscopy but asymptomatic bacteriuria was uncommon after these procedures in either group. Recurrent infections were related to age, sex and treatment center. They were common in boys and girls entering under 1 year of age but were less common in girls and rare in boys entering after 1 year of age. Recurrences were lowest among the Finnish children and highest in the German and Belgian children.


Renal scars and parenchymal thinning in children with vesicoureteral reflux: a 5-year report of the International Reflux Study in Children (European branch). A total of 321 children less than 11 years old with nonobstructive grade III or IV vesicoureteral reflux and with previous urinary tract infection was randomly allocated to medical or surgical treatment in the European branch of the International Reflux Study in Children. (Randomization was stratified for age, sex, grade of reflux, presence of renal scarring, interval since last urinary tract infection and treating hospital). The results of excretory urography are reported for 233 girls and 73 boys treated according to the random allocation, 89% of whom were followed for 5 years. After 5 years in the medical group (155 children) new renal scars were seen in 19 and new renal parenchymal thinning in 11. The proportions were almost identical among 151 children allocated to surgical treatment with 20 new scars and 15 new thinnings. Progression of established scars was also similar in both groups. However, the new scars developed sooner after surgery than during medical treatment. In 6 surgically treated children postoperative obstruction was followed by the development of new scars. In addition, 12 patients showed new scars approximately 6 months after successful surgery, while in only 2 children scars developed more than 6 months after surgery. In 11 children of the medical group new scars were seen more than 6 months after allocation. More new scars developed in the children with parenchymal thinning at entry (23%) than in those with scarred or normal kidneys at entry (10% each) (p < 0.05). The younger the patients at entry, the higher the frequency of new scars (less than 2 years 19.8%) 2 to 4 years 9.8% and 5 years or more 4.6%, p < 0.05).


Surgical results in the International Reflux Study in Children (Europe). In the European part of the International Reflux Study in Children (7 participating centers) 151 infants and children were randomly allocated to surgical treatment of primary grades III and IV vesicoureteral reflux. Reimplantation was performed unilaterally in 65 patients and bilaterally in 86, for a total of 237 ureters reimplanted. The patients were followed at regular intervals for 5 years. Reflux was absent in 231 of the reimplanted ureters (97.5%) at the end of 5 years. No patient underwent reoperation for reflux. In 10 ureters (4.2%, 10 patients) obstruction developed postoperatively and 7 needed reoperation. All reoperations were performed on the left side. Of the obstructed kidneys new scars developed in 6 during the 5-year followup. Including these cases, the number of new renal scars was equal in the surgical and medical groups (20 each). The number of pyelonephritic episodes during followup was significantly less in the surgical group (without chemoprophylaxis) than in the medical group (on chemoprophylaxis). No kidneys were lost and no child became hypertensive. If voiding cystourethrography and excretory urography were normal 6 months postoperatively, the reflux had been permanently eradicated and postoperative obstruction could be ruled out. In this study the patients who underwent reimplantation had a 74% (112 of 151) chance of an uncomplicated postoperative course (no persisting reflux, obstruction, pyelonephritis or severe renal damage).