Hidrodistensión bajo anestesia más inyección de Onabotulinumtoxin A en pacientes con síndrome de dolor vesical refractario a tratamiento conservador / Hydrodistension plus Onabotulinumtoxin A in bladder pain syndrome refractory to conservative treatments

Introducción: En el síndrome de dolor vesical (SDV) refractario a tratamientos conservadores, la guía europea contempla la hidrodistensión (HD) vesical bajo anestesia y la inyección de Onabotulinumtoxin A (OnabotA) de manera conjunta. El objetivo fue evaluar nuestra experiencia en la aplicación de la técnica. Material y métodos: Estudio prospectivo de 25 pacientes con SDV sometidos a HD más inyección submucosa de 100 U de OnabotA en trígono. Las lesiones de Hunner fueron tratadas endoscópicamente mediante resección o electrocoagulación. Se realizaron 38 procedimientos (25 primeras intervenciones y 13 reintervenciones). Para estudiar la modificación clínica se evaluó la mejoría subjetiva (escalas TBS y PGIC), la escala visual analógica (EVA) para dolor, el cuestionario BPIC-SS y el diario miccional de 3 días. Para el análisis de datos se emplearon los test de Wilcoxon, Kruskal-Wallis, Kaplan-Meier y Log-Rank. Resultados: Observamos mejoría subjetiva en 21 pacientes (84%), en 47% de ellos mejoría importante, en 41,2% moderada y en 11,8% leve. No hubo mejoría en 4 pacientes. Se objetivó una reducción postratamiento en la EVA de dolor (de 7,1 a 1,8 puntos; p = 0,001), en la frecuencia miccional diurna (de 11,8 a 7,5; p = 0,012) y nocturna (de 5,9 a 3,6; p = 0,003) y en el cuestionario BPIC-SS (de 27,9 a 11,2 puntos; p = 0,042). El grado de mejoría no tuvo relación con la edad, con la presencia de lesiones vesicales ni con el tratamiento de las recaídas. La mediana en la duración de la mejoría fue de 7 meses (IC 95%: 5,69-8,31) de manera global, aunque en los pacientes menores de 65 años fue algo mayor. Se produjeron complicaciones leves en el 23,7% de los casos. Conclusiones: La realización conjunta de HD más inyección de OnabotA es una opción terapéutica válida en el SDV refractario, con buenos resultados clínicos y manteniendo la efectividad en los retratamientos

Introduction: For bladder pain syndrome (BPS) refractory to conservative treatment, the European guidelines consider bladder hydrodistention (HD) under anaesthesia and the injection of Onabotulinumtoxin A (OnabotA) jointly. The objective of this study was to assess our experience in implementing this technique. Material and methods: A prospective study of 25 patients with BPS who underwent HD plus a submucosal injection of 100 U of OnabotA in trigone. The Hunner lesions were treated endoscopically using resection or electrocoagulation. Thirty-eight procedures were performed (25 first interventions and 13 reoperations). To study the clinical change, we evaluated the subjective improvement (Treatment Benefit Scale [TBS] and Patient Global Impression of Change [PGIC] scales), the visual analogue scale (VAS) for pain, the Bladder Pain/Interstitial Cystitis Symptom Score (BPIC-SS) questionnaire and the voiding diary for 3 days. For the data analysis, we employed the Wilcoxon, Kruskal-Wallis, Kaplan-Meier and log-rank tests. Results: We observed subjective improvement in 21 patients (84%), which was significant in 47% of these patients, moderate in 41.2% and slight in 11.8%. Four patients did not improve. A post-treatment reduction in the pain VAS (from 7.1 to 1.8 points; P = .001), in daytime (from 11.8 to 7.5; P = .012) and night-time (from 5.9 to 3.6; P = .003) voiding frequency and in the BPIC-SS (from 27.9 to 11.2 points;P = .042). The degree of improvement was not related to age, the presence of bladder lesions or the treatment of relapses. The median duration of improvement was 7 months (95% CI 5.69-8.31), although this duration was somewhat longer for the patients younger than 65 years. Mild complications occurred in 23.7% of the cases. Conclusions: The joint implementation of HD plus OnabotA is a valid therapeutic option in refractory BPS, which provides good clinical results and maintains its effectiveness in retreatments

Hydrodistension plus Onabotulinumtoxin A in bladder pain syndrome refractory to conservative treatments. / Hidrodistensión bajo anestesia más inyección de Onabotulinumtoxin A en pacientes con síndrome de dolor vesical refractario a tratamiento conservador.

INTRODUCTION: For bladder pain syndrome (BPS) refractory to conservative treatment, the European guidelines consider bladder hydrodistention (HD) under anaesthesia and the injection of Onabotulinumtoxin A (OnabotA) jointly. The objective of this study was to assess our experience in implementing this technique. MATERIAL AND METHODS: A prospective study of 25 patients with BPS who underwent HD plus a submucosal injection of 100 U of OnabotA in trigone. The Hunner lesions were treated endoscopically using resection or electrocoagulation. Thirty-eight procedures were performed (25 first interventions and 13 reoperations). To study the clinical change, we evaluated the subjective improvement (Treatment Benefit Scale [TBS] and Patient Global Impression of Change [PGIC] scales), the visual analogue scale (VAS) for pain, the Bladder Pain/Interstitial Cystitis Symptom Score (BPIC-SS) questionnaire and the voiding diary for 3 days. For the data analysis, we employed the Wilcoxon, Kruskal-Wallis, Kaplan-Meier and log-rank tests. RESULTS: We observed subjective improvement in 21 patients (84%), which was significant in 47% of these patients, moderate in 41.2% and slight in 11.8%. Four patients did not improve. A post-treatment reduction in the pain VAS (from 7.1 to 1.8 points; P=.001), in daytime (from 11.8 to 7.5; P=.012) and night-time (from 5.9 to 3.6; P=.003) voiding frequency and in the BPIC-SS (from 27.9 to 11.2 points; P=.042). The degree of improvement was not related to age, the presence of bladder lesions or the treatment of relapses. The median duration of improvement was 7 months (95% CI 5.69-8.31), although this duration was somewhat longer for the patients younger than 65 years. Mild complications occurred in 23.7% of the cases. CONCLUSIONS: The joint implementation of HD plus OnabotA is a valid therapeutic option in refractory BPS, which provides good clinical results and maintains its effectiveness in retreatments.

The activity of hsp90 alpha promoter is regulated by NF-kappa B transcription factors.

Heat-shock proteins (HSP) 90 exert a relevant role in the survival and response to therapy of many neoplastic cell types. Here, we show that the promoter of hsp90alpha gene, that encodes the inducible form of HSP90, is regulated by nuclear factor-kappaB (NF-kappaB) activity. Indeed, we found that NF-kappaB factors bound to one of the two putative consensus sequences present in the hsp90alpha-flanking region; mutation of such motif hampered the phorbol-myristate-13-acetate-stimulated expression of a luciferase reporter gene under the control of the hsp90alpha promoter. Furthermore, the downmodulation of NF-kappaB (p65) levels by a specific small interfering (si) RNA resulted in reducing the levels of endogenous HSP90alpha protein. These findings disclose a previously unrecognized mechanism that contributes to connect NF-kappaB factors and HSPs in cell defence machinery.

An homologue of the human 100-kDa protein (p100) is differentially expressed by Histoplasma capsulatum during infection of murine macrophages.

Using differential display reverse transcription-PCR (DDRT-PCR) we have identified several sequences that are specifically expressed by Histoplasma capsulatum during infection of murine macrophages (MPhi). Here, we report the characterization of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of MPhi. Screening of a cDNA library of H. capsulatum allowed us to isolate a clone, pHc12-E, that contains the complete coding sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Analysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respectively 30 and 33% identity with human and Caenorhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein consists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose three-dimensional (3D) structure is related to staphylococcal thermonuclease, followed by a modified fifth "hybrid" domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes.

Sugar oxidoreductases and veratryl alcohol oxidase as related to lignin degradation.

Properties of cellobiose:quinone oxidoreductase (CBQ), cellobiose dehydrogenase (CDH), glyoxal oxidase (GLOX), glucose oxidases and veratryl alcohol oxidase (VAO) are reviewed. There is strong evidence that CDH reduces quinones, phenoxy and cation radicals. Glucose oxidases (glucose 1-oxidase and pyranose 2-oxidase) and VAO have been less investigated but evidence for reduction of the above compounds is accumulating. Pyranose oxidase, glyoxal oxidase and VAO are very important for hydrogen peroxide production by white-rot fungi. CDH is only produced on cellulose or on wood, whereas pyranose oxidase and VAO are produced both on wood and on rich glucose media suggesting that the lignin degrading white-rot fungi may use different quinone and radical reducing enzymes to regulate lignin polymerization/depolymerization depending on the substrate and cultivation conditions. Intracellular quinone reductases are also produced. Whether brown-rot fungi in general produce CBQ/CDH or VAO is not clear. The Fe(III) reducing ability of both CDH and certain phenolate compounds agree with the rapid depolymerization of cellulose by brown-rot fungi. The interaction of Fe(III) reduction with the hydrogen peroxide producing system in white-rot and brown-rot fungi requires more investigation.

The gene, protein and glycan structures of laccase from Pleurotus ostreatus.

A member of the laccase multigene family in Pleurotus ostreatus has been cloned and sequenced. The gene structure has been determined by comparison with the corresponding cDNA, synthesized by reverse transcription/PCR amplification. The gene encode a laccase isoenzyme of 533 amino acids which has already been purified and characterized [Palmieri, G., Giardina, P., Marzullo, L., Desiderio, B., Nitti, G., Cannio, R. & Sannia, G.(1993) Appl. Microbiol. Biotechnol. 39, 632-636]. More than 92% of the protein sequence, including the N and C termini, has been verified by fast-atom-bombardment mass spectrometry, thus confirming the correspondence between the gene and its protein product. The protein was N-glycosylated Asn444. Glycan analysis showed the presence of only a high-mannose structure containing varying numbers of mannose residues. The presence of O-linked oligosaccharides as well as other post-translational modification could be ruled out by the mass analysis.

Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus.

The gene (pox1) encoding a phenol oxidase from Pleurotus ostreatus, a lignin-degrading basidiomycete, was cloned and sequenced, and the corresponding pox1 cDNA was also synthesized and sequenced. The isolated gene consists of 2,592 bp, with the coding sequence being interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA consensus sequences could be identified at positions -174 and -84, respectively. The isolation of a second cDNA (pox2 cDNA), showing 84% similarity, and of the corresponding truncated genomic clones demonstrated the existence of a multigene family coding for isoforms of laccase in P. ostreatus. PCR amplifications of specific regions on the DNA of isolated monokaryons proved that the two genes are not allelic forms. The POX1 amino acid sequence deduced was compared with those of other known laccases from different fungi.

Veratryl alcohol oxidase from Pleurotus ostreatus participates in lignin biodegradation and prevents polymerization of laccase-oxidized substrates.

Oxidative enzymes (laccases and peroxidases) isolated from the culture media of different fungi are involved in the basic mechanism of ligninolysis via radical intermediates. However, experiments aimed at reproducing natural biodegradation in vitro have been unsuccessful so far since the single biocatalysts alone are not able to solubilize lignins because of the simultaneous recondensation of these intermediates. FAD oxidases can prevent this side reaction in lignin depolymerization by reducing quinonoids and radical compounds. This study investigates the possible role of a laccase and a FAD-dependent aryl alcohol oxidase (veratryl alcohol oxidase, VAO) excreted by the basidiomycete Pleurotus ostreatus. In fact, we found that VAO is able to reduce synthetic quinones, laccase-generated quinonoids, and phenoxy radicals with concomitant oxidation of veratryl alcohol to veratryl aldehyde. This cooperative action of laccase and VAO also prevented the polymerization of phenolic compounds and reduced the molecular weight of soluble lignosulfonates to a significant extent.

A new enzyme immobilization procedure using copper alginate gel: application to a fungal phenol oxidase.

A new procedure was developed for enzyme immobilization by entrapment in copper alginate gel. The mechanical properties of the copper alginate gel were characterized and compared with those of the most widely used calcium alginate. The system was applied to the immobilization of a fungal phenol oxidase. Optimal conditions for enzyme immobilization were set up: the system immobilized 85% of the enzyme, and the remaining 15% was recovered in the aqueous immobilization medium. The stability and activity of the immobilized enzyme were studied. After immobilization, the enzyme was active in a wider pH range, the temperature of its optimal activity was shifted to lower values, and the possibility of storage at 4 degrees C was greatly improved. The immobilized enzyme generally increased the rate of oxidation of various substrates. The results indicate a potential use of this system for the construction of bioreactors to be used in the detoxification of polluted waste waters.

Stability and activity of a phenol oxidase from the ligninolytic fungus Pleurotus ostreatus.

Three different phenol oxidases produced by the basidiomycete fungus Pleurotus ostreatus have been isolated and their main structural, enzymatic and physico-chemical properties characterized. Studies have focused on the most abundantly secreted of these proteins, a copper-enzyme specific towards ortho-diphenol substrates. This protein was purified to homogeneity and part of its primary structure determined by direct protein sequencing. The influence of pH, temperature and presence of water-soluble or water-insoluble organic solvents on the activity and stability of the enzyme were also investigated. These data can be used for applying bioreactors to problems of environmental concern such as waste-water treatment.

Genetic recombination in the alpha 2 domain of the E alpha chain yields an Ed molecule with altered T cell activation.

We have used a novel T cell selection strategy to isolate a mutant of an H-2d/f murine macrophage line defective in its ability to present antigen to some Ed-restricted helper T cells. This mutant has an amino acid substitution in the alpha 2 domain of the Ed molecule. The mutation changes the sequence at codon 177 from ACC to CAC, which results in a threonine to histidine substitution and appears to be the first in vitro mutation to have arisen by genetic recombination. Even though the mutation is distal to the proposed antigen-binding groove, it affects antigen presentation, presumably by altering the scaffolding for the antigen-binding groove. This type of mutant might not be readily isolated using other selection techniques.

Infusion of cholecystokinin between meals into free-feeding rats fails to prolong the intermeal interval.

The sulfated octapeptide of cholecystokinin (CCK-8) was infused intraperitoneally into 7 free-feeding male Sprague Dawley rats over a 6-day period. Infusions were given near the end of each free-feeding meal (1.87 microgram/meal/rat), and also during the intermeal interval in gradually increasing doses (0.10-0.63 microgram/5 min/rat). Food intake was continuously monitored and the infusions were controlled by microcomputer. Meal patterns, total food intake, and body weights during drug infusion were compared with data collected during a baseline period when only saline was infused. Meal-contingent CCK-8 infusion produced a significant 29.9% decrease in meal size which persisted throughout the drug-infusion period. Intermeal infusion of CCK-8 failed to prolong the intermeal interval (IMI) but it did initially prevent the compensatory decrease in IMI and increased feeding frequency expected after meal size was reduced. By the last day of drug infusion, total daily food intake recovered to baseline levels due to increased feeding frequency. Body weight was only transiently reduced by CCK-8 infusion. These findings show that tolerance does not develop to the action of CCK-8 to suppress meal size, and the administration of exogenous CCK-8 to free-feeding rats does not persistently prolong the intermeal interval.